Methods of isolating T cells and T cell receptors having antigenic specificity for a cancer-specific mutation from peripheral blood

ABSTRACT

Disclosed are methods of isolating T cells and TCRs having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. Also disclosed are related methods of preparing a population of cells, populations of cells, TCRs, pharmaceutical compositions, and methods of treating or preventing cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national stage of PCT/US2016/030137,filed Apr. 29, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 62/155,830, filed May 1, 2015, each of which isincorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberZIABC010984 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 98,375 Byte ASCII (Text) file named“730741_ST25.TXT” dated Oct. 16, 2017.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL)or cells that have been genetically engineered to express an anti-cancerantigen T cell receptor (TCR) can produce positive clinical responses insome cancer patients. Nevertheless, obstacles to the successful use ofACT for the widespread treatment of cancer and other diseases remain.For example, T cells and TCRs that specifically recognize cancerantigens may be difficult to identify and/or isolate from a patient.Accordingly, there is a need for improved methods of obtainingcancer-reactive T cells and TCRs.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of isolating T cellshaving antigenic specificity for a mutated amino acid sequence encodedby a cancer-specific mutation, the method comprising obtaining a bulkpopulation of peripheral blood mononuclear cells (PBMCs) from a sampleof peripheral blood from a patient; selecting T cells that expressprogrammed cell death 1 (PD-1) from the bulk population; separating theT cells that express PD-1 from cells that do not express PD-1 to obtaina T cell population enriched for T cells that express PD-1; identifyingone or more genes in the nucleic acid of a cancer cell of the patient,each gene containing a cancer-specific mutation that encodes a mutatedamino acid sequence; inducing autologous antigen presenting cells (APCs)of the patient to present the mutated amino acid sequence; co-culturingT cells from the population enriched for T cells that express PD-1 withthe autologous APCs that present the mutated amino acid sequence; andselecting the T cells that (a) were co-cultured with the autologous APCsthat present the mutated amino acid sequence and (b) have antigenicspecificity for the mutated amino acid sequence presented in the contextof a major histocompatibility complex (MHC) molecule expressed by thepatient.

Another embodiment of the invention provides a method of isolating Tcells having antigenic specificity for a mutated amino acid sequenceencoded by a cancer-specific mutation, the method comprising obtaining afirst population of PBMCs from a sample of peripheral blood from apatient; selecting T cells that express PD-1 from the bulk population;separating the T cells that express PD-1 from cells that do not expressPD-1 to obtain a T cell population enriched for T cells that expressPD-1; isolating nucleotide sequence(s) that encode(s) one or moreTCR(s), or antigen-binding portion(s) thereof, from the T cells of thepopulation enriched for T cells that express PD-1; introducing thenucleotide sequence(s) encoding the TCR(s), or antigen bindingportion(s) thereof, into further population(s) of PBMCs to obtain Tcells that express the TCR(s), or antigen binding portion(s) thereof;identifying one or more genes in the nucleic acid of a cancer cell ofthe patient, each gene containing a cancer-specific mutation thatencodes a mutated amino acid sequence; inducing autologous APCs of thepatient to present the mutated amino acid sequence; co-culturing the Tcells that express the TCR(s), or antigen binding portion(s) thereof,with the autologous APCs that present the mutated amino acid sequence;and selecting the T cells that (a) were co-cultured with the autologousAPCs that present the mutated amino acid sequence and (b) have antigenicspecificity for the mutated amino acid sequence presented in the contextof a MHC molecule expressed by the patient.

Another embodiment of the invention provides an isolated or purified TCRcomprising the amino acid sequences of (a) SEQ ID NOs: 5-10; (b) SEQ IDNOs: 13-18; (c) SEQ ID NOs: 21-26; (d) SEQ ID NOs: 29-34; or (e) SEQ IDNOs: 37-42.

Another embodiment of the invention provides an isolated or purifiedpolypeptide comprising the amino acid sequences of (a) SEQ ID NOs: 5-10;(b) SEQ ID NOs: 13-18; (c) SEQ ID NOs: 21-26; (d) SEQ ID NOs: 29-34; or(e) SEQ ID NOs: 37-42.

An isolated or purified protein comprising (a) a first polypeptide chaincomprising the amino acid sequences of SEQ ID NOs: 5-7 and a secondpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:8-10; (b) a first polypeptide chain comprising the amino acid sequencesof SEQ ID NOs: 13-15 and a second polypeptide chain comprising the aminoacid sequences of SEQ ID NOs: 16-18; (c) a first polypeptide chaincomprising the amino acid sequences of SEQ ID NOs: 21-23 and a secondpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:24-26; (d) a first polypeptide chain comprising the amino acid sequencesof SEQ ID NOs: 29-31 and a second polypeptide chain comprising the aminoacid sequences of SEQ ID NOs: 32-34; or (e) a first polypeptide chaincomprising the amino acid sequences of SEQ ID NOs: 37-39 and a secondpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:40-42.

Additional embodiments of the invention provide related nucleic acids,recombinant expression vectors, host cells, populations of cells,pharmaceutical compositions, and methods of treating or preventingcancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a graph showing the frequency of 4-1BB+ cells (%) in thepopulations of peripheral blood lymphocytes (PBL) transduced with acontrol (empty) vector (Vector Td) or a TCR isolated from tandemminigene (TMG)-1 specific cells isolated from PD-1hi population (Vb3 TCRTd) cultured alone (unshaded bars) or upon co-culture with OKT3 antibody(grey bars) or target autologous dendritic cells pulsed with no peptide(vertically striped bars), wild type CASP8 (wt CASP8) peptide (checkeredbars), or mutated CASP8 (mut CASP8) peptide (diagonally striped bars).

FIG. 2A is a graph showing the reactivity (as determined by 4-1BBupregulation on CD3+CD8+ cells) of retrovirally transduced lymphocytesfrom subject NCI-3998 expressing MAGEA6_(E>K), PDS5A_(Y>F;H>Y), andMED13_(P>S)-specific TCRs against the autologous tumor cell line 3998mel.

FIG. 2B is a graph showing the reactivity of the circulating CD8+PD-1−and CD8+PD-1+ lymphocytes against their autologous tumor cell line.Frequency of 4-1BB on CD3+ cells is shown (mean±SEM).

FIG. 2C is a graph showing the TCRB overlap between the tumor-residentCD8⁺PD-1⁺ cells, and the blood-derived CD8³⁰, CD8⁺PD-1⁻, and CD8⁺PD-1⁺cells. TCRB overlap of 1 indicates 100% similarity between twopopulations. n.s. not significant, **P<0.01 using Dunn's test formultiple comparisons.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a method of isolating T cellshaving antigenic specificity for a mutated amino acid sequence encodedby a cancer-specific mutation. The invention provides many advantages.For example, the inventive methods may, advantageously, obtain cancerantigen-reactive T cells from a patient's peripheral blood, which is amore accessible and abundant source of T cells as compared to othertissues such as, for example, tumor. By obtaining cancerantigen-reactive T cells from the peripheral blood, the inventivemethods may, advantageously, obtain cancer antigen-reactive T cellswithout using invasive techniques such as, for example, surgery orbiopsy, which may be required when obtaining T cells from other tissuessuch as, for example, a tumor. Cancer antigen-reactive T cells are notfrequently found in the peripheral blood. Nevertheless, the inventivemethods overcome this obstacle, and effectively and efficiently identifyand enrich for these infrequent, cancer antigen-reactive T cells fromthe peripheral blood. In addition, the inventive methods make itpossible to administer ACT to patients that have no tumors available forTIL harvest. The inventive methods may also reduce the cost of ACT,making ACT available for a larger number of patients.

Moreover, the inventive methods may rapidly assess a large number ofmutations restricted by all of the patient's MHC molecules at one time,which may identify the full repertoire of the patient'smutation-reactive T cells. Additionally, by distinguishing immunogeniccancer mutations from (a) silent cancer-specific mutations (which do notencode a mutated amino acid sequence) and (b) cancer-specific mutationsthat encode a non-immunogenic amino acid sequence, the inventive methodsmay identify one or more cancer-specific, mutated amino acid sequencesthat may be targeted by a T cell, a TCR, or an antigen-binding portionthereof. The mutated amino acid sequences could be used to synthesizepeptides and immunize patients to treat or prevent cancer recurrence. Inaddition, the invention may provide T cells, TCRs, and antigen-bindingportions thereof, having antigenic specificity for mutated amino acidsequences encoded by cancer-specific mutations that are unique to thepatient, thereby providing “personalized” T cells, TCRs, andantigen-binding portions thereof, that may be useful for treating orpreventing the patient's cancer. The inventive methods may also avoidthe technical biases inherent in traditional methods of identifyingcancer antigens such as, for example, those using cDNA libraries, andmay also be less time-consuming and laborious than those methods. Forexample, the inventive methods may select mutation-reactive T cellswithout co-culturing the T cells with tumor cell lines, which may bedifficult to generate, particularly for e.g., epithelial cancers.Without being bound to a particular theory or mechanism, it is believedthat the inventive methods may identify and isolate T cells and TCRs, orantigen-binding portions thereof, that target the destruction of cancercells while minimizing or eliminating the destruction of normal,non-cancerous cells, thereby reducing or eliminating toxicity.Accordingly, the invention may also provide T cells, TCRs, orantigen-binding portions thereof, that successfully treat or preventcancer such as, for example, cancers that do not respond to other typesof treatment such as, for example, chemotherapy alone, surgery, orradiation.

The method may comprise obtaining a bulk population of PBMCs from asample of peripheral blood of a patient by any suitable method known inthe art. Suitable methods of obtaining a bulk population of PBMCs mayinclude, but are not limited to, a blood draw and/or a leukopheresis.The bulk population of PBMCs obtained from a peripheral blood sample maycomprise T cells, including tumor-reactive T cells.

The method may comprise selecting T cells that express PD-1 from thebulk population. In an embodiment of the invention, the T cells thatexpress PD-1 may be PD-1hi cells. In a preferred embodiment, selecting Tcells that express PD-1 from the bulk population comprises selecting Tcells that co-express (a) PD-1 and (b) any one or more of CD3, CD4, CD8,T cell immunoglobulin and mucin domain 3 (TIM-3), and CD27. In anembodiment of the invention, the cells that express CD3, CD4, CD8,TIM-3, or CD27 may be CD3hi, CD4hi, CD8hi, TIM-3hi, or CD27hi cells,respectively. The method may comprise specifically selecting the cellsin any suitable manner. Preferably, the selecting is carried out usingflow cytometry. The flow cytometry may be carried out using any suitablemethod known in the art. The flow cytometry may employ any suitableantibodies and stains. For example, the specific selection of PD-1, CD3,CD4, CD8, TIM-3, or CD27 may be carried out using anti-PD-1, anti-CD3,anti-CD4, anti-CD8, anti-TIM-3, or anti-CD27 antibodies, respectively.Preferably, the antibody is chosen such that it specifically recognizesand binds to the particular biomarker being selected. The antibody orantibodies may be conjugated to a bead (e.g., a magnetic bead) or to afluorochrome. Preferably, the flow cytometry is fluorescence-activatedcell sorting (FACS).

In an embodiment of the invention, selecting may comprise specificallyselecting PD-1+ T cells that are also positive for expression of (i) anyone of CD4, CD8, TIM-3, and CD27; (ii) both of CD8 and TIM-3; (iii) bothof CD8 and CD27; (iv) both of TIM-3 and CD27; (v) all three of CD8,TIM-3, and CD27; (vi) both of CD4 and TIM-3; (vii) both of CD4 and CD27;or (viii) all three of CD4, TIM-3, and CD27. In another embodiment ofthe invention, any one or more of the populations of (i)-(viii) may alsoco-express CD3.

In an embodiment of the invention, selecting T cells that express PD-1from the bulk population comprises selecting any one or more of (a)CD8+PD-1+; (b) PD-1+TIM-3+; (c) PD-1+CD27+; (d) CD8+PD-1 hi; (e)CD8+PD-1+TIM-3+; (f) CD8+PD-1+CD27hi; (g) CD8+PD-1+CD27+; (h)CD8+PD-1+TIM-3−; (i) CD8+PD-1+CD27−; (j) CD4+PD-1+; (k) CD4+PD-1hi; (1)CD4+PD-1+TIM-3+; (m) CD4+PD-1+CD27hi; (n) CD4+PD-1+CD27+; (o)CD4+PD-1+TIM-3−; and (p) CD4+PD-1+CD27− T cells. In another embodimentof the invention, any one or more of the populations of (a)-(p) may alsoco-express CD3.

As used herein, the term “positive” (which may be abbreviated as “+”),with reference to expression of the indicated cell marker, means thatthe cell expresses the indicated cell marker at any detectable level,which may include, for example, expression at a low (but detectable)level as well as expression at a high (hi) level. The term “negative”(which may be abbreviated as “−”), as used herein with reference toexpression of the indicated cell marker, means that the cell does notexpress the indicated cell marker at a detectable level. The term “high”(which may be abbreviated as “hi”), as used herein with reference toexpression of the indicated cell marker, refers to a subset of cellsthat are positive for expression of the indicated cell marker whichstain more brightly for the indicated cell marker using one of thefollowing methods (e.g., FACS, flow cytometry, immunofluorescence assaysor microscopy) than other cells that are positive for expression of theindicated cell marker. For example, cells with a “high” level ofexpression of the indicated cell marker may stain more brightly thanabout 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, ora range of any two of the foregoing values, of the other cells that arepositive for expression of the indicated cell marker.

In an embodiment of the invention, selecting T cells that express PD-1may comprise selecting combinations of PD-1⁺ cells, each PD-1+ cellexpressing any one, two, or more different markers as described herein.In this regard, the method may produce a cell population that isenriched for tumor-reactive cells that comprises a mixture of PD-1+cells, each PD-1+ cell expressing any one, two, or more differentmarkers described herein. In an embodiment of the invention, selecting Tcells that express PD-1 comprises selecting a combination of (i) bothPD-1−CD8+ cells and PD-1+TIM-3+ cells; (ii) both PD-1+CD8+ cells andPD-1+CD27+ cells; (iii) both PD-1+TIM-3+ cells and PD-1+CD27+ cells;(iv) all of PD-1+CD8+ cells, PD-1+TIM-3+ cells, and PD-1+CD27+ cells;(v) both PD-1+CD4+ cells and PD-1+TIM-3+ cells; (vi) both PD-1+CD4+cells and PD-1+CD27+ cells; (vii) all of PD-1+CD4+ cells, PD-1+TIM-3+cells, and PD-1+CD27+ cells, or (viii) a combination of any of thepopulations of (i)-(vii). In another embodiment of the invention, anyone or more of the populations of (i)-(vii) may also co-express CD3. Inanother embodiment of the invention, selecting T cells that express PD-1comprises selecting a combination of any two or more of (a) CD8+PD-1+;(b) PD-1+TIM-3+; (c) PD-1+CD27+; (d) CD8+PD-1hi; (e) CD8+PD-1+TIM-3+;(f) CD8+PD-1+CD27hi; (g) CD8+PD-1+CD27+; (h) CD8+PD-1+TIM-3−; (i)CD8+PD-1+CD27−; (j) CD4+PD-1+; (k) CD4+PD-1hi; (1) CD4+PD-1+TIM-3+; (m)CD4+PD-1+CD27hi; (n) CD4+PD-1+CD27+; (o) CD4+PD-1+TIM-3−; and (p)CD4+PD-1+CD27− cells. In another embodiment of the invention, any one ormore of the populations of (a)-(p) may also co-express CD3.

The method may comprise separating the T cells that express PD-1 fromcells that do not express PD-1 to obtain a T cell population enrichedfor T cells that express PD-1. In this regard, the selected cells may bephysically separated from unselected cells, i.e., the cells that do notexpress PD-1. The selected cells may be separated from unselected cellsby any suitable method such as, for example, sorting.

The method may comprise identifying one or more genes in the nucleicacid of a cancer cell of a patient, each gene containing acancer-specific mutation that encodes a mutated amino acid sequence. Thecancer cell may be obtained from any bodily sample derived from apatient which contains or is expected to contain tumor or cancer cells.The bodily sample may be any tissue sample such as blood, a tissuesample obtained from the primary tumor or from tumor metastases, or anyother sample containing tumor or cancer cells. The nucleic acid of thecancer cell may be DNA or RNA.

In order to identify cancer-specific mutations, the method may furthercomprise sequencing nucleic acid such as DNA or RNA of normal,noncancerous cells and comparing the nucleic acid sequence of the cancercell with the sequence of the normal, noncancerous cell. The normal,noncancerous cell may be obtained from the patient or a differentindividual.

The cancer-specific mutation may be any mutation in any gene whichencodes a mutated amino acid sequence (also referred to as a “non-silentmutation”) and which is expressed in a cancer cell but not in a normal,noncancerous cell. Non-limiting examples of cancer-specific mutationsthat may be identified in the inventive methods include missense,nonsense, insertion, deletion, duplication, frameshift, and repeatexpansion mutations. In an embodiment of the invention, the methodcomprises identifying at least one gene containing a cancer-specificmutation which encodes a mutated amino acid sequence. However, thenumber of genes containing such a cancer-specific mutation that may beidentified using the inventive methods is not limited and may includemore than one gene (for example, about 2, about 3, about 4, about 5,about 10, about 11, about 12, about 13, about 14, about 15, about 20,about 25, about 30, about 40, about 50, about 60, about 70, about 80,about 90, about 100, about 150, about 200, about 400, about 600, about800, about 1000, about 1500, about 2000 or more, or a range defined byany two of the foregoing values). Likewise, in an embodiment of theinvention, the method comprises identifying at least one cancer-specificmutation which encodes a mutated amino acid sequence. However, thenumber of such cancer-specific mutations that may be identified usingthe inventive methods is not limited and may include more than onecancer-specific mutation (for example, about 2, about 3, about 4, about5, about 10, about 11, about 12, about 13, about 14, about 15, about 20,about 25, about 30, about 40, about 50, about 60, about 70, about 80,about 90, about 100, about 150, about 200, about 400, about 600, about800, about 1000, about 1500, about 2000 or more, or a range defined byany two of the foregoing values). In an embodiment in which more thanone cancer-specific mutation is identified, the cancer-specificmutations may be located in the same gene or in different genes.

In an embodiment, identifying one or more genes in the nucleic acid of acancer cell comprises sequencing the whole exome, the whole genome, orthe whole transcriptome of the cancer cell. Sequencing may be carriedout in any suitable manner known in the art. Examples of sequencingtechniques that may be useful in the inventive methods include NextGeneration Sequencing (NGS) (also referred to as “massively parallelsequencing technology”) or Third Generation Sequencing. NGS refers tonon-Sanger-based high-throughput DNA sequencing technologies. With NGS,millions or billions of DNA strands may be sequenced in parallel,yielding substantially more throughput and minimizing the need for thefragment-cloning methods that are often used in Sanger sequencing ofgenomes. In NGS, nucleic acid templates may be randomly read in parallelalong the entire genome by breaking the entire genome into small pieces.NGS may, advantageously, provide nucleic acid sequence information of awhole genome, exome, or transcriptome in very short time periods, e.g.,within about 1 to about 2 weeks, preferably within about 1 to about 7days, or most preferably, within less than about 24 hours. Multiple NGSplatforms which are commercially available or which are described in theliterature can be used in the context of the inventive methods, e.g.,those described in Zhang et al., J. Genet. Genomics, 38(3): 95-109(2011) and Voelkerding et al., Clinical Chemistry, 55: 641-658 (2009).

Non-limiting examples of NGS technologies and platforms includesequencing-by-synthesis (also known as “pyrosequencing”) (asimplemented, e.g., using the GS-FLX 454 Genome Sequencer, 454 LifeSciences (Branford, Conn.), ILLUMINA SOLEXA Genome Analyzer (IlluminaInc., San Diego, Calif.), or the ILLUMINA HISEQ 2000 Genome Analyzer(Illumina), or as described in, e.g., Ronaghi et al., Science,281(5375): 363-365 (1998)), sequencing-by-ligation (as implemented,e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad,Calif.) or the POLONATOR G.007 platform (Dover Systems, Salem, N.H.)),single-molecule sequencing (as implemented, e.g., using the PACBIO RSsystem (Pacific Biosciences (Menlo Park, Calif.) or the HELISCOPEplatform (Helicos Biosciences (Cambridge, Mass.)), nano-technology forsingle-molecule sequencing (as implemented, e.g., using the GRIDONplatform of Oxford Nanopore Technologies (Oxford, UK), thehybridization-assisted nano-pore sequencing (HANS) platforms developedby Nabsys (Providence, R.I.), and the ligase-based DNA sequencingplatform with DNA nanoball (DNB) technology referred to as probe-anchorligation (cPAL)), electron microscopy-based technology forsingle-molecule sequencing, and ion semiconductor sequencing.

The method may comprise inducing autologous APCs of the patient topresent the mutated amino acid sequence. The APCs may include any cellswhich present peptide fragments of proteins in association with MHCmolecules on their cell surface. The APCs may include, for example, anyone or more of macrophages, dendritic cells (DCs), langerhans cells,B-lymphocytes, and T-cells. Preferably, the APCs are DCs. By usingautologous APCs from the patient, the inventive methods may,advantageously, identify T cells, TCRs, and antigen-binding portionsthereof, that have antigenic specificity for a mutated amino acidsequence encoded by a cancer-specific mutation that is presented in thecontext of an MHC molecule expressed by the patient. The MHC moleculecan be any MHC molecule expressed by the patient including, but notlimited to, MHC Class I, MHC Class II, HLA-A, HLA-B, HLA-C, HLA-DM,HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules. The inventive methods may,advantageously, identify mutated amino acid sequences presented in thecontext of any MHC molecule expressed by the patient without using, forexample, epitope prediction algorithms to identify MHC molecules ormutated amino acid sequences, which may be useful only for a select fewMHC class I alleles and may be constrained by the limited availabilityof reagents to select mutation-reactive T cells (e.g., an incomplete setof MHC tetramers). Accordingly, in an embodiment of the invention, theinventive methods advantageously identify mutated amino acid sequencespresented in the context of any MHC molecule expressed by the patientand are not limited to any particular MHC molecule. Preferably, theautologous APCs are antigen-negative autologous APCs.

Inducing autologous APCs of the patient to present the mutated aminoacid sequence may be carried out using any suitable method known in theart. In an embodiment of the invention, inducing autologous APCs of thepatient to present the mutated amino acid sequence comprises pulsing theautologous APCs with peptides comprising the mutated amino acid sequenceor a pool of peptides, each peptide in the pool comprising a differentmutated amino acid sequence. Each of the mutated amino acid sequences inthe pool may be encoded by a gene containing a cancer specific mutation.In this regard, the autologous APCs may be cultured with a peptide or apool of peptides comprising the mutated amino acid sequence in a mannersuch that the APCs internalize the peptide(s) and display the mutatedamino acid sequence(s), bound to an MHC molecule, on the cell membrane.In an embodiment in which more than one gene is identified, each genecontaining a cancer-specific mutation that encodes a mutated amino acidsequence, the method may comprise pulsing the autologous APCs with apool of peptides, each peptide in the pool comprising a differentmutated amino acid sequence. Methods of pulsing APCs are known in theart and are described in, e.g., Solheim (Ed.), Antigen Processing andPresentation Protocols (Methods in Molecular Biology), Human Press,(2010). The peptide(s) used to pulse the APCs may include the mutatedamino acid(s) encoded by the cancer-specific mutation. The peptide(s)may further comprise any suitable number of contiguous amino acids fromthe endogenous protein encoded by the identified gene on each of thecarboxyl side and the amino side of the mutated amino acid(s). Thenumber of contiguous amino acids from the endogenous protein flankingeach side of the mutation is not limited and may be, for example, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, about 20, or a range defined by any two of the foregoingvalues. Preferably, the peptide(s) comprise(s) about 12 contiguous aminoacids from the endogenous protein on each side of the mutated aminoacid(s).

In an embodiment of the invention, inducing autologous APCs of thepatient to present the mutated amino acid sequence comprises introducinga nucleotide sequence encoding the mutated amino acid sequence into theAPCs. The nucleotide sequence is introduced into the APCs so that theAPCs express and display the mutated amino acid sequence, bound to anMHC molecule, on the cell membrane. The nucleotide sequence encoding themutated amino acid may be RNA or DNA. Introducing a nucleotide sequenceinto APCs may be carried out in any of a variety of different ways knownin the art as described in, e.g., Solheim et al. supra. Non-limitingexamples of techniques that are useful for introducing a nucleotidesequence into APCs include transformation, transduction, transfection,and electroporation. In an embodiment in which more than one gene isidentified, the method may comprise preparing more than one nucleotidesequence, each encoding a mutated amino acid sequence encoded by adifferent gene, and introducing each nucleotide sequence into adifferent population of autologous APCs. In this regard, multiplepopulations of autologous APCs, each population expressing anddisplaying a different mutated amino acid sequence, may be obtained.

In an embodiment in which more than one gene is identified, each genecontaining a cancer-specific mutation that encodes a mutated amino acidsequence, the method may comprise introducing a nucleotide sequenceencoding the more than one gene. In this regard, in an embodiment of theinvention, the nucleotide sequence introduced into the autologous APCsis a tandem minigene (TMG) construct, each minigene comprising adifferent gene, each gene including a cancer-specific mutation thatencodes a mutated amino acid sequence. Each minigene may encode onemutation identified by the inventive methods flanked on each side of themutation by any suitable number of contiguous amino acids from theendogenous protein encoded by the identified gene, as described hereinwith respect to other aspects of the invention. The number of minigenesin the construct is not limited and may include for example, about 5,about 10, about 11, about 12, about 13, about 14, about 15, about 20,about 25, or more, or a range defined by any two of the foregoingvalues. The APCs express the mutated amino acid sequences encoded by theTMG construct and display the mutated amino acid sequences, bound to anMHC molecule, on the cell membranes. In an embodiment, the method maycomprise preparing more than one TMG construct, each construct encodinga different set of mutated amino acid sequences encoded by differentgenes, and introducing each TMG construct into a different population ofautologous APCs. In this regard, multiple populations of autologousAPCs, each population expressing and displaying mutated amino acidsequences encoded by different TMG constructs, may be obtained.

The method may comprise co-culturing T cells from the populationenriched for T cells that express PD-1 with the autologous APCs thatpresent the mutated amino acid sequence. The T cells from the populationenriched for T cells that express PD-1 are obtained from peripheralblood as described herein with respect to other aspects of theinvention. The T cells can express PD-1 and any of the other cellmarkers described herein with respect to other aspects of the invention.The method may comprise co-culturing the T cells that express PD-1 andautologous APCs so that the T cells encounter the mutated amino acidsequence presented by the APCs in such a manner that the T cellsspecifically bind to and immunologically recognize a mutated amino acidsequence presented by the APCs. In an embodiment of the invention, the Tcells are co-cultured in direct contact with the autologous APCs.

The method may comprise selecting the T cells that (a) were co-culturedwith the autologous APCs that present the mutated amino acid sequenceand (b) have antigenic specificity for the mutated amino acid sequencepresented in the context of a MHC molecule expressed by the patient. Thephrase “antigenic specificity,” as used herein, means that a T cell,TCR, or the antigen-binding portion thereof, expressed by the T cell,can specifically bind to and immunologically recognize the mutated aminoacid sequence encoded by the cancer-specific mutation. The selecting maycomprise identifying the T cells that have antigenic specificity for themutated amino acid sequence and separating them from T cells that do nothave antigenic specificity for the mutated amino acid sequence.Selecting the T cells having antigenic specificity for the mutated aminoacid sequence may be carried out in any suitable manner. In anembodiment of the invention, the method comprises expanding the numbersof T cells that express PD-1, e.g., by co-culturing with a T cell growthfactor, such as interleukin (IL)-2 or IL-15, or as described herein withrespect to other aspects of the invention, prior to selecting the Tcells that have antigenic specificity for the mutated amino acidsequence. In an embodiment of the invention, the method does notcomprise expanding the numbers of T cells that express PD-1 with a Tcell growth factor, such as IL-2 or IL-15 prior to selecting the T cellsthat have antigenic specificity for the mutated amino acid sequence.

For example, upon co-culture of the T cells that express PD-1 with theAPCs that present the mutated amino acid sequence, T cells havingantigenic specificity for the mutated amino acid sequence may expressany one or more of a variety of T cell activation markers which may beused to identify those T cells having antigenic specificity for themutated amino acid sequence. Such T cell activation markers may include,but are not limited to, PD-1, lymphocyte-activation gene 3 (LAG-3),TIM-3, 4-1BB, OX40, and CD107a. Accordingly, in an embodiment of theinvention, selecting the T cells that have antigenic specificity for themutated amino acid sequence comprises selecting the T cells that expressany one or more of PD-1, LAG-3, TIM-3, 4-1BB, OX40, and CD107a. Cellsexpressing one or more T cell activation markers may be sorted on thebasis of expression of the marker using any of a variety of techniquesknown in the art such as, for example, FACS or magnetic-activated cellsorting (MACS) as described in, e.g., Turcotte et al., Clin. CancerRes., 20(2): 331-43 (2013) and Gros et al., J. Clin. Invest., 124(5):2246-59 (2014).

In another embodiment of the invention, selecting the T cells that haveantigenic specificity for the mutated amino acid sequence comprisesselecting the T cells (i) that secrete a greater amount of one or morecytokines upon co-culture with APCs that present the mutated amino acidsequence as compared to the amount of the one or more cytokines secretedby a negative control or (ii) in which at least twice as many of thenumbers of T cells secrete one or more cytokines upon co-culture withAPCs that present the mutated amino acid sequence as compared to thenumbers of negative control T cells that secrete the one or morecytokines. The one or more cytokines may comprise any cytokine thesecretion of which by a T cell is characteristic of T cell activation(e.g., a TCR expressed by the T cells specifically binding to andimmunologically recognizing the mutated amino acid sequence).Non-limiting examples of cytokines, the secretion of which ischaracteristic of T cell activation, include IFN-γ, IL-2, and tumornecrosis factor alpha (TNF-α), granulocyte/monocyte colony stimulatingfactor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

For example, the T cells may be considered to have “antigenicspecificity” for the mutated amino acid sequence if the T cells secreteat least twice as much IFN-γ upon co-culture with (a) antigen-negativeAPCs pulsed with a concentration of a peptide comprising the mutatedamino acid sequence (e.g., about 0.001 ng/mL to about 10 μg/mL, e.g.,0.001 ng/ml, 0.005 ng/mL, 0.01 ng/ml, 0.05 ng/ml, 0.1 ng/mL, 0.5 ng/mL,1 ng/mL, 5 ng/mL, 100 ng/mL, 1 μg/mL, 5 μg/mL, or 10 μg/mL) or (b) APCsinto which a nucleotide sequence encoding the mutated amino acidsequence has been introduced as compared to the amount of IFN-γ secretedby a negative control. The negative control may be, for example,autologous T cells (e.g., derived from PBMCs) co-cultured with (a)antigen-negative APCs pulsed with the same concentration of anirrelevant peptide (e.g., the wild-type amino acid sequence, or someother peptide with a different sequence from the mutated amino acidsequence) or (b) APCs into which a nucleotide sequence encoding anirrelevant peptide sequence has been introduced. The T cells may alsohave “antigenic specificity” for the mutated amino acid sequence if theT cells secrete a greater amount of IFN-γ upon co-culture withantigen-negative APCs pulsed with higher concentrations of a peptidecomprising the mutated amino acid sequence as compared to a negativecontrol, for example, the negative control described above. IFN-γsecretion may be measured by methods known in the art such as, forexample, enzyme-linked immunosorbent assay (ELISA).

Alternatively or additionally, the T cells may be considered to have“antigenic specificity” for the mutated amino acid sequence if at leasttwice as many of the numbers of T cells secrete IFN-γ upon co-culturewith (a) antigen-negative APCs pulsed with a concentration of a peptidecomprising the mutated amino acid sequence or (b) APCs into which anucleotide sequence encoding the mutated amino acid sequence has beenintroduced as compared to the numbers of negative control T cells thatsecrete IFN-γ. The concentration of peptide and the negative control maybe as described herein with respect to other aspects of the invention.The numbers of cells secreting IFN-γ may be measured by methods known inthe art such as, for example, ELISPOT.

While T cells having antigenic specificity for the mutated amino acidsequence may both (1) express any one or more T cells activation markersdescribed herein and (2) secrete a greater amount of one or morecytokines as described herein, in an embodiment of the invention, Tcells having antigenic specificity for the mutated amino acid sequencemay express any one or more T cell activation markers without secretinga greater amount of one or more cytokines or may secrete a greateramount of one or more cytokines without expressing any one or more Tcell activation markers.

In another embodiment of the invention, selecting the T cells that haveantigenic specificity for the mutated amino acid sequence comprisesselectively growing the T cells that have antigenic specificity for themutated amino acid sequence. In this regard, the method may compriseco-culturing the T cells with autologous APCs in such a manner as tofavor the growth of the T cells that have antigenic specificity for themutated amino acid sequence over the T cells that do not have antigenicspecificity for the mutated amino acid sequence. Accordingly, apopulation of T cells is provided that has a higher proportion of Tcells that have antigenic specificity for the mutated amino acidsequence as compared to T cells that do not have antigenic specificityfor the mutated amino acid sequence.

In an embodiment of the invention in which T cells are co-cultured withautologous APCs expressing multiple mutated amino acid sequences (e.g.,multiple mutated amino acid sequences encoded by a TMG construct ormultiple mutated amino acid sequences in a pool of peptides pulsed ontoautologous APCs), selecting the T cells may further comprise separatelyassessing T cells for antigenic specificity for each of the multiplemutated amino acid sequences. For example, the inventive method mayfurther comprise separately inducing autologous APCs of the patient topresent each mutated amino acid sequence encoded by the construct (orincluded in the pool), as described herein with respect to other aspectsof the invention (for example, by providing separate APC populations,each presenting a different mutated amino acid sequence encoded by theconstruct (or included in the pool)). The method may further compriseseparately co-culturing T cells with the different populations ofautologous APCs that present each mutated amino acid sequence, asdescribed herein with respect to other aspects of the invention. Themethod may further comprise separately selecting the T cells that (a)were co-cultured with the autologous APCs that present the mutated aminoacid sequence and (b) have antigenic specificity for the mutated aminoacid sequence presented in the context of a MHC molecule expressed bythe patient, as described herein with respect to other aspects of theinvention. In this regard, the method may comprise determining whichmutated amino acid sequence encoded by a TMG construct that encodesmultiple mutated amino acid sequences (or included in the pool) areimmunologically recognized by the T cells (e.g., by process ofelimination).

The method may further comprise isolating a nucleotide sequence thatencodes the TCR, or the antigen-binding portion thereof, from theselected T cells, wherein the TCR, or the antigen-binding portionthereof, has antigenic specificity for the mutated amino acid sequenceencoded by the cancer-specific mutation. In an embodiment of theinvention, prior to isolating the nucleotide sequence that encodes theTCR, or the antigen-binding portion thereof, the numbers selected Tcells that have antigenic specificity for the mutated amino acidsequence may be expanded. Expansion of the numbers of T cells can beaccomplished by any of a number of methods as are known in the art asdescribed in, for example, U.S. Pat. Nos. 8,034,334; 8,383,099; U.S.Patent Application Publication No. 2012/0244133; Dudley et al., J.Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods,128:189-201 (1990). In an embodiment, expansion of the numbers of Tcells is carried out by culturing the T cells with OKT3 antibody, IL-2,and feeder PBMC (e.g., irradiated allogeneic PBMC). In anotherembodiment of the invention, the numbers of selected T cells that haveantigenic specificity for the mutated amino acid sequence are notexpanded prior to isolating the nucleotide sequence that encodes theTCR, or the antigen-binding portion thereof. For example, the TCR, orantigen binding portion thereof, may be isolated from a single cell.

The “the antigen-binding portion” of the TCR, as used herein, refers toany portion comprising contiguous amino acids of the TCR of which it isa part, provided that the antigen-binding portion specifically binds tothe mutated amino acid sequence encoded by the gene identified asdescribed herein with respect to other aspects of the invention. Theterm “antigen-binding portion” refers to any part or fragment of the TCRof the invention, which part or fragment retains the biological activityof the TCR of which it is a part (the parent TCR). Antigen-bindingportions encompass, for example, those parts of a TCR that retain theability to specifically bind to the mutated amino acid sequence, ordetect, treat, or prevent cancer, to a similar extent, the same extent,or to a higher extent, as compared to the parent TCR. In reference tothe parent TCR, the functional portion can comprise, for instance, about10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR.

The antigen-binding portion can comprise an antigen-binding portion ofeither or both of the α and β chains of the TCR of the invention, suchas a portion comprising one or more of the complementarity determiningregion (CDR)1, CDR2, and CDR3 of the variable region(s) of the α chainand/or β chain of the TCR of the invention. In an embodiment of theinvention, the antigen-binding portion can comprise the amino acidsequence of the CDR1 of the α chain (CDR1α), the CDR2 of the α chain(CDR2α), the CDR3 of the α chain (CDR3α), the CDR1 of the β chain(CDR1β), the CDR2 of the β chain (CDR2β), the CDR3 of the β chain(CDR3β), or any combination thereof. Preferably, the antigen-bindingportion comprises the amino acid sequences of CDR1α, CDR2α, and CDR3α;the amino acid sequences of CDR1β, CDR2β, and CDR3β; or the amino acidsequences of all of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of theinventive TCR.

In an embodiment of the invention, the antigen-binding portion cancomprise, for instance, the variable region of the inventive TCRcomprising a combination of the CDR regions set forth above. In thisregard, the antigen-binding portion can comprise the amino acid sequenceof the variable region of the α chain (Vα), the amino acid sequence ofthe variable region of the β chain (Vβ), or the amino acid sequences ofboth of the Vα and Vβ of the inventive TCR.

In an embodiment of the invention, the antigen-binding portion maycomprise a combination of a variable region and a constant region. Inthis regard, the antigen-binding portion can comprise the entire lengthof the α or β chain, or both of the α and β chains, of the inventiveTCR.

Isolating the nucleotide sequence that encodes the TCR, or theantigen-binding portion thereof, from the selected T cells may becarried out in any suitable manner known in the art. For example, themethod may comprise isolating RNA from the selected T cells andsequencing the TCR, or the antigen-binding portion thereof, usingestablished molecular cloning techniques and reagents such as, forexample, 5′ Rapid Amplification of cDNA Ends (RACE) polymerase chainreaction (PCR) using TCR-α and -β chain constant primers.

In an embodiment of the invention, the method may comprise cloning thenucleotide sequence that encodes the TCR, or the antigen-binding portionthereof, into a recombinant expression vector using establishedmolecular cloning techniques as described in, e.g., Green et al. (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 4th Ed. (2012). For purposes herein, the term “recombinantexpression vector” means a genetically-modified oligonucleotide orpolynucleotide construct that permits the expression of an mRNA,protein, polypeptide, or peptide by a host cell, when the constructcomprises a nucleotide sequence encoding the mRNA, protein, polypeptide,or peptide, and the vector is contacted with the cell under conditionssufficient to have the mRNA, protein, polypeptide, or peptide expressedwithin the cell. The vectors of the invention are notnaturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. The recombinant expression vectors can comprise anytype of nucleotides, including, but not limited to DNA (e.g.,complementary DCA (cDNA)) and RNA, which can be single-stranded ordouble-stranded, synthesized or obtained in part from natural sources,and which can contain natural, non-natural or altered nucleotides. Therecombinant expression vectors can comprise naturally-occurring,non-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the non-naturally occurring or altered nucleotidesor internucleotide linkages does not hinder the transcription orreplication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting oftransposon/transposase, the pUC series (Fennentas Life Sciences), thepBluescript series (Stratagene, La Jolla, Calif.), the pET series(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophagevectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149,also can be used. Examples of plant expression vectors include pBI01,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animalexpression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).Preferably, the recombinant expression vector is a viral vector, e.g., aretroviral vector.

The TCR, or the antigen-binding portion thereof, isolated by theinventive methods may be useful for preparing cells for adoptive celltherapies. In this regard, an embodiment of the invention provides amethod of preparing a population of cells that express a TCR, or anantigen-binding portion thereof, having antigenic specificity for amutated amino acid sequence encoded by a cancer-specific mutation, themethod comprising isolating a TCR, or an antigen-binding portionthereof, as described herein with respect to other aspects of theinvention, and introducing the nucleotide sequence encoding the isolatedTCR, or the antigen-binding portion thereof, into host cells to obtaincells that express the TCR, or the antigen-binding portion thereof.

Introducing the nucleotide sequence (e.g., a recombinant expressionvector) encoding the isolated TCR, or the antigen-binding portionthereof, into host cells may be carried out in any of a variety ofdifferent ways known in the art as described in, e.g., Green et al.supra. Non-limiting examples of techniques that are useful forintroducing a nucleotide sequence into host cells includetransformation, transduction, transfection, and electroporation.

The host cell into which the nucleotide sequence encoding the TCR, orantigen binding portion thereof, is introduced may be any type of cellthat can contain the inventive recombinant expression vector. The hostcell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, orcan be a prokaryotic cell, e.g., bacteria or protozoa. The host cell canbe a cultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell, can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5α E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH59α cell. For purposes of producing the TCR,or antigen binding portion thereof, the host cell is preferably amammalian cell. Most preferably, the host cell is a human cell. Whilethe host cell can be of any cell type, can originate from any type oftissue, and can be of any developmental stage, the host cell preferablyis a PBL or a PBMC. More preferably, the host cell is a T cell.

In an embodiment of the invention, the PBMC include T cells. The T cellsmay be any type of T cell. Without being bound to a particular theory ormechanism, it is believed that less differentiated, “younger” T cellsmay be associated with any one or more of greater in vivo persistence,proliferation, and antitumor activity as compared to moredifferentiated, “older” T cells. Accordingly, the inventive methods may,advantageously, identify and isolate a TCR, or an antigen-bindingportion thereof, that has antigenic specificity for the mutated aminoacid sequence and introduce the TCR, or an antigen-binding portionthereof, into “younger” T cells that may provide any one or more ofgreater in vivo persistence, proliferation, and antitumor activity ascompared to “older” T cells (e.g., effector cells in a patient's tumor).

In an embodiment of the invention, the host cells are autologous to thepatient. In this regard, the TCRs, or the antigen-binding portionsthereof, identified and isolated by the inventive methods may bepersonalized to each patient. However, in another embodiment, theinventive methods may identify and isolate TCRs, or the antigen-bindingportions thereof, that have antigenic specificity against a mutatedamino acid sequence that is encoded by a recurrent (also referred to as“hot-spot”) cancer-specific mutation. In this regard, the method maycomprise introducing the nucleotide sequence encoding the isolated TCR,or the antigen-binding portion thereof, into host cells that areallogeneic to the patient. For example, the method may compriseintroducing the nucleotide sequence encoding the isolated TCR, or theantigen-binding portion thereof, into the host cells from anotherpatient whose tumors express the same mutation in the context of thesame MHC molecule.

In an embodiment of the invention, the method further comprisesexpanding the numbers of host cells that express the TCR, or theantigen-binding portion thereof. The numbers of host cells may beexpanded, for example, as described herein with respect to other aspectsof the invention. In this regard, the inventive methods may,advantageously, generate a large number of T cells having antigenicspecificity for the mutated amino acid sequence.

Another embodiment of the invention provides a TCR, or anantigen-binding portion thereof, isolated by any of the methodsdescribed herein with respect to other aspects of the invention. Anembodiment of the invention provides a TCR comprising two polypeptides(i.e., polypeptide chains), such as an alpha (α) chain of a TCR, a beta(β) chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of aTCR, or a combination thereof. Another embodiment of the inventionprovides an antigen-binding portion of the TCR comprising one or moreCDR regions, one or more variable regions, or one or both of the α and βchains of the TCR, as described herein with respect to other aspects ofthe invention. The polypeptides of the inventive TCR, or theantigen-binding portion thereof, can comprise any amino acid sequence,provided that the TCR, or the antigen-binding portion thereof, hasantigenic specificity for the mutated amino acid sequence encoded by thecancer-specific mutation.

In an embodiment of the invention, the TCR, or antigen binding portionthereof, has antigenic specificity for MAGE-A6_(E168K). The phrase“antigenic specificity,” as used herein, means that the TCR canspecifically bind to and immunologically recognize the particularantigen under discussion. Wild-type, non-mutated MAGE-A6 comprises theamino acid sequence of SEQ ID NO: 74. MAGE-A6_(E168K) comprises theamino acid sequence of SEQ ID NO: 74 except that the glutamic acid atposition 168 of SEQ ID NO: 74 is substituted with lysine. In anembodiment of the invention, the TCR has antigenic specificity for theMAGE-A6_(E168K) amino acid sequence of SEQ ID NO: 77.

The anti-MAGE-A6_(E168K) TCR, or antigen binding portion thereof,comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 5 or13 (CDR1 of α chain), a CDR2 comprising the amino acid sequence of SEQID NO: 6 or 14 (CDR2 of α chain), and a CDR3 comprising the amino acidsequence of SEQ ID NO: 7 or 15 (CDR3 of α chain), and a secondpolypeptide chain comprising a CDR1 comprising the amino acid sequenceof SEQ ID NO: 8 or 16 (CDR1 of β chain), a CDR2 comprising the aminoacid sequence of SEQ ID NO: 9 or 17 (CDR2 of β chain), and a CDR3comprising the amino acid sequence of SEQ ID NO: 10 or 18 (CDR3 of βchain). In this regard, the inventive TCR, or antigen binding portionthereof, can comprise any one or more of the amino acid sequencesselected from the group consisting of SEQ ID NOs: 5-10 and SEQ ID NOs:13-18. In an especially preferred embodiment, the TCR, or antigenbinding portion thereof, comprises the amino acid sequences of (i) allof SEQ ID NOs: 5-10 or (ii) all of SEQ ID NOs: 13-18.

In an embodiment of the invention, the TCR, or antigen binding portionthereof, has antigenic specificity for PDS5A_(Y1000F; H1007Y).Wild-type, non-mutated PDS5A comprises the amino acid sequence of SEQ IDNO: 75. PDS5A_(Y1000F; H1007Y) comprises the amino acid sequence of SEQID NO: 75 except that the tyrosine at position 1000 of SEQ ID NO: 75 issubstituted with phenylalanine and the histidine at position 1007 of SEQID NO: 75 is substituted with tyrosine. In an embodiment of theinvention, the TCR, or antigen binding portion thereof, has antigenicspecificity for the PDS5A_(Y1000F; H1007Y) amino acid sequence of SEQ IDNO: 78.

In an embodiment of the invention, the anti-PDS5A_(Y1000F; H1007Y) TCR,or antigen binding portion thereof comprises the amino acid sequence ofSEQ ID NO: 21 (CDR1 of α chain), a CDR2 comprising the amino acidsequence of SEQ ID NO: 22 (CDR2 of α chain), and a CDR3 comprising theamino acid sequence of SEQ ID NO: 23 (CDR3 of α chain), and a secondpolypeptide chain comprising a CDR1 comprising the amino acid sequenceof SEQ ID NO: 24 (CDR1 β chain), a CDR2 comprising the amino acidsequence of SEQ ID NO: 25 (CDR2 of β chain), and a CDR3 comprising theamino acid sequence of SEQ ID NO: 26 (CDR3 of β chain). In this regard,the inventive TCR, or antigen binding portion thereof, can comprise anyone or more of the amino acid sequences selected from the groupconsisting of SEQ ID NOs: 21-26. In an especially preferred embodiment,the TCR, or antigen binding portion thereof, comprises the amino acidsequences of all of SEQ ID NOs: 21-26.

In an embodiment of the invention, the TCR, or antigen binding portionthereof, has antigenic specificity for MED13_(P1691S). Wild-type,non-mutated MED13 comprises the amino acid sequence of SEQ ID NO: 76.MED13_(P1691S) comprises the amino acid sequence of SEQ ID NO: 76 exceptthat the proline at position 1691 of SEQ ID NO: 76 is substituted withserine. In an embodiment of the invention, the TCR, or antigen bindingportion thereof, has antigenic specificity for the MED13_(P1691S) aminoacid sequence of SEQ ID NO: 79.

In an embodiment of the invention, the anti-MED13_(P1691S) TCR, orantigen binding portion thereof, comprises a CDR1 comprising the aminoacid sequence of SEQ ID NO: 29 or 37 (CDR1 of α chain), a CDR2comprising the amino acid sequence of SEQ ID NO: 30 or 38 (CDR2 of αchain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31or 39 (CDR3 of α chain), and a second polypeptide chain comprising aCDR1 comprising the amino acid sequence of SEQ ID NO: 32 or 40 (CDR1 ofβ chain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 33 or41 (CDR2 of β chain), and a CDR3 comprising the amino acid sequence ofSEQ ID NO: 34 or 42 (CDR3 of β chain). In this regard, the inventiveTCR, or antigen binding portion thereof, can comprise any one or more ofthe amino acid sequences selected from the group consisting of SEQ IDNOs: 29-34 and SEQ ID NOs: 37-42. In an especially preferred embodiment,the TCR, or antigen binding portion thereof, comprises the amino acidsequences of (i) all of SEQ ID NOs: 29-34 or (ii) all of SEQ ID NOs:37-42.

In an embodiment of the invention, the TCR can comprise an amino acidsequence of a variable region of a TCR comprising the CDRs set forthabove. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 11 or 19 (the variable region of an α chain of ananti-MAGE-A6_(E168K) TCR); SEQ ID NO: 12, wherein X at position 2 of SEQID NO: 12 is Gly or Ala (the variable region of a β chain of ananti-MAGE-A6_(E168K) TCR); SEQ ID NO: 20, wherein X at position 2 of SEQID NO: 20 is Gly or Ala (the variable region of a β chain of ananti-MAGE-A6_(E168K) TCR); both SEQ ID NOs: 11 and 12; both SEQ ID NOs:19 and 20; SEQ ID NO: 27 (the variable region of an α chain of theanti-PDS5A_(Y1000F; H1007Y)TCR); SEQ ID NO: 28, wherein X at position 2of SEQ ID NO: 28 is Gly or Ala (the variable region of a β chain of theanti-PDS5A_(Y1000F; H1007Y) TCR); both SEQ ID NOs: 27 and 28; SEQ ID NO:35 or 43 (the variable region of an α chain of an anti-MED13_(P1691S)TCR); SEQ ID NO: 36, wherein X at position 2 of SEQ ID NO: 36 is Gly orAla (the variable region of a β chain of an anti-MED13_(P1691S) TCR);SEQ ID NO: 44, wherein X at position 2 of SEQ ID NO: 44 is Gly or Ala(the variable region of a β chain of an anti-MED13_(P1691S) TCR); bothSEQ ID NOs: 35 and 36; or both SEQ ID NOs: 43 and 44. Preferably, theinventive TCR comprises the amino acid sequences of (a) SEQ ID NOs:11-12; (b) SEQ ID NOs: 19-20; (c) SEQ ID NOs: 27-28; (d) SEQ ID NOs:35-36; or (e) SEQ ID NOs: 43-44.

The inventive TCRs may further comprise a constant region derived fromany suitable species such as, e.g., human or mouse. As used herein, theterm “murine” or “human,” when referring to a TCR or any component of aTCR described herein (e.g., complementarity determining region (CDR),variable region, constant region, alpha chain, and/or beta chain), meansa TCR (or component thereof) which is derived from a mouse or a human,respectively, i.e., a TCR (or component thereof) that originated from orwas, at one time, expressed by a mouse T cell or a human T cell,respectively.

In an embodiment of the invention, the constant region is a humanconstant region. In this regard, the TCR can comprise SEQ ID NO: 61(constant region of a human α chain); SEQ ID NO: 62 (constant region ofa human β chain); SEQ ID NO: 63 (constant region of a human β chain);both SEQ ID NO: 61 and SEQ ID NO: 62; or both SEQ ID NOs: 61 and 63. TheTCR may comprise any of the CDR regions as described herein with respectto other aspects of the invention. In another embodiment of theinvention, the TCR may comprise any of the variable regions describedherein with respect to other aspects of the invention.

In an embodiment of the invention, the TCR further comprises a murineconstant region. For example, the TCR may be a chimeric TCR comprising ahuman variable region and a murine constant region. In this regard, theTCR can comprise SEQ ID NO: 47 (constant region of a murine α chain);SEQ ID NO: 48 (constant region of a murine β chain); or both SEQ ID NO:47 and SEQ ID NO: 48. The chimeric TCR may comprise any of the CDRregions as described herein with respect to other aspects of theinvention. In another embodiment of the invention, the chimeric TCR maycomprise any of the variable regions described herein with respect toother aspects of the invention. In an embodiment of the invention, theTCR comprises a murine constant region, optionally with one, two, three,or four amino acid substitution(s) in the constant region of one or bothof the alpha and beta chains, as described herein with respect to otheraspects of the invention. In an embodiment of the invention, the TCRcomprises a murine constant region, optionally with one, two, three, orfour amino acid substitution(s) in the murine constant region of thealpha chain and one amino acid substitution in the murine constantregion of the beta chain, as described herein with respect to otheraspects of the invention.

In some embodiments, the TCRs comprising the substituted amino acidsequence(s) advantageously provide one or more of increased recognitionof mutated amino acid sequence-positive targets, increased expression bya host cell, and increased anti-tumor activity as compared to the parentTCR comprising an unsubstituted amino acid sequence. In general, thesubstituted amino acid sequences of the murine constant regions of theTCR α and β chains, SEQ ID NOs: 45 and 46, respectively, correspond withall or portions of the unsubstituted murine constant region amino acidsequences SEQ ID NOs: 47 and 48, respectively, with SEQ ID NO: 45 havingone, two, three, or four amino acid substitution(s) when compared to SEQID NO: 47 and SEQ ID NO: 46 having one amino acid substitution whencompared to SEQ ID NO: 48. In this regard, an embodiment of theinvention provides a TCR comprising the amino acid sequences of one orboth of (a) SEQ ID NO: 45 (constant region of alpha chain), wherein (i)X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala,Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met,Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (b) SEQ IDNO: 46 (constant region of beta chain), wherein X at position 57 is Seror Cys. In an embodiment of the invention, the TCR comprising SEQ ID NO:45 does not comprise SEQ ID NO: 47 (unsubstituted murine constant regionof alpha chain). In an embodiment of the invention, the TCR comprisingSEQ ID NO: 46 does not comprise SEQ ID NO: 48 (unsubstituted murineconstant region of beta chain).

In an embodiment of the invention, the substituted amino acid sequenceincludes cysteine substitutions in the constant region of one or both ofthe α and β chains to provide a cysteine-substituted TCR. Opposingcysteines in the α and the β chains provide a disulfide bond that linksthe constant regions of the α and the β chains of the substituted TCR toone another and which is not present in a TCR comprising theunsubstituted human constant region or the unsubstituted murine constantregion. In this regard, the TCR is a cysteine-substituted TCR in whichone or both of the native Thr48 of SEQ ID NO: 47 and the native Ser57 ofSEQ ID NO: 48 may be substituted with Cys. Preferably, both of thenative Thr48 of SEQ ID NO: 47 and the native Ser57 of SEQ ID NO: 48 aresubstituted with Cys. In an embodiment, the cysteine-substituted TCRcomprises an alpha chain constant region comprising the amino acidsequence of SEQ ID NO: 45, wherein X at position 48 is Cys, X atposition 112 is the native Ser, X at position 114 is the native Met, andX at position 115 is the native Gly, and a beta chain constant regioncomprising the amino acid sequence of SEQ ID NO: 46, wherein X atposition 57 is Cys. The cysteine-substituted TCRs of the invention mayinclude the substituted constant region in addition to any of the CDRsor variable regions described herein.

In an embodiment of the invention, the substituted amino acid sequenceincludes substitutions of one, two, or three amino acids in thetransmembrane (TM) domain of the constant region of one or both of the αand β chains with a hydrophobic amino acid to provide a hydrophobicamino acid-substituted TCR. The hydrophobic amino acid substitution(s)in the TM domain of the TCR may increase the hydrophobicity of the TMdomain of the TCR as compared to a TCR that lacks the hydrophobic aminoacid substitution(s) in the TM domain. In this regard, the TCR may be ahydrophobic amino acid-substituted TCR in which one, two, or three ofthe native Ser112, Met114, and Gly115 of SEQ ID NO: 47 may,independently, be substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe,Met, or Trp; preferably with Leu, Ile, or Val. Preferably, all three ofthe native Ser112, Met114, and Gly115 of SEQ ID NO: 47 are,independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met,or Trp; preferably with Leu, Ile, or Val. In an embodiment, thehydrophobic amino acid-substituted TCR comprises an alpha chain constantregion comprising the amino acid sequence of SEQ ID NO: 45, wherein X atposition 48 is the native Thr, X at position 112 is Ser, Gly, Ala, Val,Leu, Ile, Pro, Phe, Met, or Trp, X at position 114 is Met, Gly, Ala,Val, Leu, Ile, Pro, Phe, Met, or Trp, and X at position 115 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Met, or Trp, and a beta chain constant regioncomprising the amino acid sequence of SEQ ID NO: 46, wherein X atposition 57 is the native Ser, wherein the hydrophobic aminoacid-substituted TCR comprising SEQ ID NO: 45 does not comprise SEQ IDNO: 47 (unsubstituted murine constant region of alpha chain).Preferably, the hydrophobic amino acid-substituted TCR comprises analpha chain constant region comprising the amino acid sequence of SEQ IDNO: 45, wherein X at position 48 is the native Thr, X at position 112 isLeu, X at position 114 is Ile, and X at position 115 is Val, and a betachain constant region comprising the amino acid sequence of SEQ ID NO:46, wherein X at position 57 is the native Ser. The hydrophobic aminoacid-substituted TCRs of the invention may include the substitutedconstant region in addition to any of the CDRs or variable regionsdescribed herein.

In an embodiment of the invention, the substituted amino acid sequenceincludes the cysteine substitutions in the constant region of one orboth of the α and β chains in combination with the substitution(s) ofone, two, or three amino acids in the transmembrane (TM) domain of theconstant region of one or both of the α and β chains with a hydrophobicamino acid (also referred to herein as “cysteine-substituted,hydrophobic amino acid-substituted TCR”). In this regard, the TCR is acysteine-substituted, hydrophobic amino acid-substituted TCR in whichthe native Thr48 of SEQ ID NO: 47 is substituted with Cys; one, two, orthree of the native Ser112, Met114, and Gly115 of SEQ ID NO: 47 are,independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met,or Trp; preferably with Leu, Ile, or Val; and the native Ser57 of SEQ IDNO: 48 is substituted with Cys. Preferably, all three of the nativeSer112, Met114, and Gly115 of SEQ ID NO: 47 are, independently,substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;preferably with Leu, Ile, or Val. In an embodiment, thecysteine-substituted, hydrophobic amino acid-substituted TCR comprisesan alpha chain constant region comprising the amino acid sequence of SEQID NO: 45, wherein X at position 48 is Cys, X at position 112 is Ser,Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, X at position 114 isMet, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and X at position115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and a beta chainconstant region comprising the amino acid sequence of SEQ ID NO: 46,wherein X at position 56 is Cys, wherein the cysteine-substituted,hydrophobic amino acid-substituted TCR comprising SEQ ID NO: 45 does notcomprise SEQ ID NO: 47 (unsubstituted murine constant region of alphachain). Preferably, the cysteine-substituted, hydrophobic aminoacid-substituted TCR comprises an alpha chain constant region comprisingthe amino acid sequence of SEQ ID NO: 49 and a beta chain constantregion comprising the amino acid sequence of SEQ ID NO: 50. Thecysteine-substituted, hydrophobic amino acid-substituted, TCRs of theinvention may include the substituted constant region in addition to anyof the CDRs or variable regions described herein. In an especiallypreferred embodiment, the cysteine-substituted, hydrophobic aminoacid-substituted TCR comprises a full-length alpha chain comprising theamino acid sequence of SEQ ID NO: 51, 53, 55, 57, or 59 and afull-length beta chain comprising the amino acid sequence of SEQ ID NO:52, 54, 56, 58, or 60. In this regard, the Cys-substituted, hydrophobicamino acid-substituted TCR can comprise the amino acid sequences of (a)both of SEQ ID NOs: 51-52; (b) both of SEQ ID NOs: 53-54; (c) both ofSEQ ID NOs: 55-56; (d) both of SEQ ID NOs: 57-58; or (e) both of SEQ IDNOs: 59-60.

Also provided by the invention is a polypeptide comprising anantigen-binding portion of any of the TCRs described herein. The term“polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds. Theantigen-binding portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent TCR.Desirably, the additional amino acids do not interfere with thebiological function of the antigen-binding portion, e.g., specificallybinding to a mutated amino acid sequence; and/or having the ability todetect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity, as compared tothe biological activity of the parent TCR.

The polypeptide can comprise an antigen-binding portion of either orboth of the α and β chains of the TCRs of the invention, such as anantigen-binding portion comprising one of more of CDR1, CDR2, and CDR3of the variable region(s) of the α chain and/or β chain of a TCR of theinvention. In an embodiment of the invention, the polypeptide cancomprise an antigen-binding portion comprising the amino acid sequenceof SEQ ID NO: 5, 13, 21, 29, or 37 (CDR1 of α chain), SEQ ID NO: 6, 14,22, 30, or 38 (CDR2 of α chain), SEQ ID NO: 7, 15, 23, 31, or 39 (CDR3of α chain), SEQ ID NO: 8, 16, 24, 32, or 40 (CDR1 of β chain), SEQ IDNO: 9, 17, 25, 33, or 41 (CDR2 of β chain), SEQ ID NO: 10, 18, 26, 34,or 42 (CDR3 of β chain), or a combination thereof. Preferably, theinventive polypeptide comprises the amino acid sequences of (a) all ofSEQ ID NOs: 5-10; (b) all of SEQ ID NOs: 13-18; (c) all of SEQ ID NOs:21-26; (d) all of SEQ ID NOs: 29-34; or (e) all of SEQ ID NOs: 37-42.

In an embodiment of the invention, the inventive polypeptide cancomprise, for instance, the variable region of the inventive TCRcomprising a combination of the CDR regions set forth above. In thisregard, the polypeptide can comprise the amino acid sequence of SEQ IDNO: 11 or 19 (the variable region of an α chain of ananti-MAGE-A6_(E168K) TCR); SEQ ID NO: 12, wherein X at position 2 of SEQID NO: 12 is Gly or Ala (the variable region of a β chain of ananti-MAGE-A6_(E168K) TCR); SEQ ID NO: 20, wherein X at position 2 of SEQID NO: 20 is Gly or Ala (the variable region of a β chain of ananti-MAGE-A6_(E168K) TCR); both SEQ ID NOs: 11 and 12; both SEQ ID NOs:19 and 20; SEQ ID NO: 27 (the variable region of an α chain of theanti-PDS5A_(Y1000F; H1007Y) TCR); SEQ ID NO: 28, wherein X at position 2of SEQ ID NO: 28 is Gly or Ala (the variable region of a β chain of theanti-PDS5A_(Y1000F; H1007Y) TCR); both SEQ ID NOs: 27 and 28; SEQ ID NO:35 or 43 (the variable region of an α chain of an anti-MED13_(P1691S)TCR); SEQ ID NO: 36, wherein X at position 2 of SEQ ID NO: 36 is Gly orAla (the variable region of a β chain of an anti-MED13_(P1691S) TCR);SEQ ID NO: 44, wherein X at position 2 of SEQ ID NO: 44 is Gly or Ala(the variable region of a β chain of an anti-MED13_(P1691S) TCR); bothSEQ ID NOs: 35 and 36; or both SEQ ID NOs: 43 and 44. Preferably, theinventive polypeptide comprises the amino acid sequences of (a) SEQ IDNOs: 11-12; (b) SEQ ID NOs: 19-20; (c) SEQ ID NOs: 27-28; (d) SEQ IDNOs: 35-36; or (e) SEQ ID NOs: 43-44.

The inventive polypeptide may further comprise a constant region derivedfrom any suitable species such as, e.g., human or mouse, describedherein or any of the substituted constant regions described herein. Inthis regard, the polypeptide can comprise the amino acid sequence of SEQID NO: 45 (constant region of α chain, substituted as described hereinwith respect to other aspects of the invention), SEQ ID NO: 47 (theunsubstituted constant region of a murine α chain), SEQ ID NO: 46(constant region of β chain, substituted as described herein withrespect to other aspects of the invention), SEQ ID NO: 48 (theunsubstituted constant region of a murine β chain), SEQ ID NO: 49(constant region of a cysteine-substituted, hydrophobic aminoacid-substituted α chain), SEQ ID NO: 50 (constant region of acysteine-substituted β chain), both SEQ ID NOs: 45 and 46, both SEQ IDNOs: 47 and 48, both SEQ ID NOs: 49 and 50, SEQ ID NO: 61 (constantregion of a human α chain); SEQ ID NO: 62 (constant region of a human βchain); SEQ ID NO: 63 (constant region of a human β chain); both SEQ IDNO: 61 and SEQ ID NO: 62; or both SEQ ID NOs: 61 and 63.

In an embodiment of the invention, the inventive polypeptide cancomprise the entire length of an α or β chain of one of the TCRsdescribed herein. In this regard, the inventive polypeptide can comprisean amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60. Preferably, the polypeptide comprises the amino acid sequences of(a) both of SEQ ID NOs: 51-52; (b) both of SEQ ID NOs: 53-54; (c) bothof SEQ ID NOs: 55-56; (d) both of SEQ ID NOs: 57-58; or (e) both of SEQID NOs: 59-60.

The invention further provides a protein comprising at least one of thepolypeptides described herein. By “protein” is meant a moleculecomprising one or more polypeptide chains.

In an embodiment of the invention, the protein may comprise the CDRsequences of the inventive TCR. In this regard, the protein of theinvention can comprise: (a) a first polypeptide chain comprising theamino acid sequences of SEQ ID NOs: 5-7 and a second polypeptide chaincomprising the amino acid sequences of: SEQ ID NOs: 8-10; (b) a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:13-15 and a second polypeptide chain comprising the amino acid sequencesof: SEQ ID NOs: 16-18; (c) a first polypeptide chain comprising theamino acid sequences of SEQ ID NOs: 21-23 and a second polypeptide chaincomprising the amino acid sequences of: SEQ ID NOs: 24-26; (d) a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:29-31 and a second polypeptide chain comprising the amino acid sequencesof: SEQ ID NOs: 32-34; or (e) a first polypeptide chain comprising theamino acid sequences of SEQ ID NOs: 37-39 and a second polypeptide chaincomprising the amino acid sequences of: SEQ ID NOs: 40-42.

In an embodiment of the invention, the protein may comprise the variableregion sequences of the inventive TCR. In this regard, the protein ofthe invention can comprise: (a) a first polypeptide chain comprising theamino acid sequence of SEQ ID NO: 11 and a second polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 12, wherein X atposition 2 of SEQ ID NO: 12 is Gly or Ala; (b) a first polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 19 and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 20,wherein X at position 2 of SEQ ID NO: 20 is Gly or Ala; (c) a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 27and a second polypeptide chain comprising the amino acid sequence of SEQID NO: 28, wherein X at position 2 of SEQ ID NO: 28 is Gly or Ala; (d) afirst polypeptide chain comprising the amino acid sequence of SEQ ID NO:35 and a second polypeptide chain comprising the amino acid sequence ofSEQ ID NO: 36, wherein X at position 2 of SEQ ID NO: 36 is Gly or Ala;or (e) a first polypeptide chain comprising the amino acid sequence ofSEQ ID NO: 43 and a second polypeptide chain comprising the amino acidsequence of SEQ ID NO: 44, wherein X at position 2 of SEQ ID NO: 44 isGly or Ala.

In an embodiment of the invention, the inventive protein may furthercomprise TCR constant region sequences. In this regard, the firstpolypeptide chain of the inventive protein may further comprise theamino acid sequence of SEQ ID NO: 45 (constant region of the alphachain, substituted as described herein with respect to other aspects ofthe invention), SEQ ID NO: 47 (the unsubstituted constant region of amurine α chain), or SEQ ID NO: 49 (constant region of acysteine-substituted, hydrophobic amino acid-substituted α chain); andthe second polypeptide chain of the inventive protein may furthercomprise the amino acid sequence of SEQ ID NO: 46 (constant region of βchain, substituted as described herein with respect to other aspects ofthe invention), SEQ ID NO: 48 (the unsubstituted constant region of amurine β chain), SEQ ID NO: 50 (constant region of acysteine-substituted β chain) SEQ ID NO: 61 (constant region of a humanα chain); SEQ ID NO: 62 (constant region of a human β chain); SEQ ID NO:63 (constant region of a human β chain); both SEQ ID NO: 61 and SEQ IDNO: 62; or both SEQ ID NOs: 61 and 63. In a preferred embodiment of theinvention, the protein comprises: (a) a first polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 45 and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 46;(b) a first polypeptide chain comprising the amino acid sequence of SEQID NO: 47 and a second polypeptide chain comprising the amino acidsequence of SEQ ID NO: 48; or (c) a first polypeptide chain comprisingthe amino acid sequence of SEQ ID NO: 49 and a second polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 50.

In an embodiment of the invention, the protein may comprise the fulllength alpha and beta chains of the inventive TCR. In this regard, theprotein may comprise (a) a first polypeptide chain comprising the aminoacid sequence of SEQ ID NO: 51 and a second polypeptide chain comprisingthe amino acid sequence of SEQ ID NO: 52; (b) a first polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 53 and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 54;(c) a first polypeptide chain comprising the amino acid sequence of SEQID NO: 55 and a second polypeptide chain comprising the amino acidsequence of SEQ ID NO: 56; (d) a first polypeptide chain comprising theamino acid sequence of SEQ ID NO: 57 and a second polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 58; or (e) a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 59and a second polypeptide chain comprising the amino acid sequence of SEQID NO: 60. In this instance, the protein of the invention can be a TCR.Alternatively, if, for example, the protein comprises a singlepolypeptide chain comprising SEQ ID NOs: 5-10; or if the first and/orsecond polypeptide chain(s) of the protein further comprise(s) otheramino acid sequences, e.g., an amino acid sequence encoding animmunoglobulin or a portion thereof, then the inventive protein can be afusion protein. In this regard, the invention also provides a fusionprotein comprising at least one of the inventive polypeptides describedherein along with at least one other polypeptide. The other polypeptidecan exist as a separate polypeptide of the fusion protein, or can existas a polypeptide, which is expressed in frame (in tandem) with one ofthe inventive polypeptides described herein. The other polypeptide canencode any peptidic or proteinaceous molecule, or a portion thereof,including, but not limited to an immunoglobulin, CD3, CD4, CD8, an MHCmolecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods.

The protein of the invention can be a recombinant antibody comprising atleast one of the inventive polypeptides described herein. As usedherein, “recombinant antibody” refers to a recombinant (e.g.,genetically engineered) protein comprising at least one of thepolypeptides of the invention and a polypeptide chain of an antibody, ora portion thereof. The polypeptide of an antibody, or portion thereof,can be a heavy chain, a light chain, a variable or constant region of aheavy or light chain, a single chain variable fragment (scFv), or an Fc,Fab, or F(ab)₂′ fragment of an antibody, etc. The polypeptide chain ofan antibody, or portion thereof, can exist as a separate polypeptide ofthe recombinant antibody. Alternatively, the polypeptide chain of anantibody, or portion thereof, can exist as a polypeptide, which isexpressed in frame (in tandem) with the polypeptide of the invention.The polypeptide of an antibody, or portion thereof, can be a polypeptideof any antibody or any antibody fragment.

The TCRs, polypeptides, and proteins of the invention can be of anylength, i.e., can comprise any number of amino acids, provided that theTCRs, polypeptides, or proteins retain their biological activity, e.g.,the ability to specifically bind to a mutated amino acid sequence;detect cancer; or treat or prevent cancer in a mammal, etc. For example,the polypeptide can be in the range of from about 50 to about 5000 aminoacids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500,600, 700, 800, 900, 1000 or more amino acids in length. In this regard,the polypeptides of the invention also include oligopeptides.

The TCRs, polypeptides, and proteins of the invention of the inventioncan comprise synthetic amino acids in place of one or morenaturally-occurring amino acids. Such synthetic amino acids are known inthe art, and include, for example, aminocyclohexane carboxylic acid,norleucine, α-amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

Included in the scope of the invention are functional variants of theinventive TCRs, polypeptides, and proteins described herein. The teen“functional variant,” as used herein, refers to a TCR, polypeptide, orprotein having substantial or significant sequence identity orsimilarity to a parent TCR, polypeptide, or protein, which functionalvariant retains the biological activity of the TCR, polypeptide, orprotein of which it is a variant. Functional variants encompass, forexample, those variants of the TCR, polypeptide, or protein describedherein (the parent TCR, polypeptide, or protein) that retain the abilityto specifically bind to a mutated amino acid sequence for which theparent TCR has antigenic specificity or to which the parent polypeptideor protein specifically binds, to a similar extent, the same extent, orto a higher extent, as the parent TCR, polypeptide, or protein. Inreference to the parent TCR, polypeptide, or protein, the functionalvariant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%,95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence to theparent TCR, polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent TCR, polypeptide, or protein with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent TCR, polypeptide, or protein with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent TCR, polypeptide, or protein.

The TCR, polypeptide, or protein can consist essentially of thespecified amino acid sequence or sequences described herein, such thatother components of the TCR, polypeptide, or protein, e.g., other aminoacids, do not materially change the biological activity of the TCR,polypeptide, or protein.

An embodiment of the invention provides a nucleic acid sequencecomprising a nucleotide sequence encoding any of the TCRs, polypeptides,or proteins described herein. “Nucleic acid” as used herein includes“polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” andgenerally means a polymer of DNA or RNA, which can be single-stranded ordouble-stranded, synthesized or obtained (e.g., isolated and/orpurified) from natural sources, which can contain natural, non-naturalor altered nucleotides, and which can contain a natural, non-natural oraltered internucleotide linkage, such as a phosphoroamidate linkage or aphosphorothioate linkage, instead of the phosphodiester found betweenthe nucleotides of an unmodified oligonucleotide. In an embodiment, thenucleic acid comprises complementary DNA (cDNA). It is generallypreferred that the nucleic acid does not comprise any insertions,deletions, inversions, and/or substitutions. However, it may be suitablein some instances, as discussed herein, for the nucleic acid to compriseone or more insertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Green and Sambrook et al., supra. For example, a nucleic acidcan be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed upon hybridization (e.g., phosphorothioate derivatives andacridine substituted nucleotides). Examples of modified nucleotides thatcan be used to generate the nucleic acids include, but are not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the TCRs, polypeptides, or proteins described herein. In anembodiment of the invention, the nucleotide sequence may comprise,consist, or consist essentially of SEQ ID NO: 64 or 66 (the variableregion of an α chain of an anti-MAGE-A6_(E168K) TCR); SEQ ID NO: 65 or67 (the variable region of a β chain of an anti-MAGE-A6_(E168K) TCR);both SEQ ID NOs: 64 and 65; both SEQ ID NOs: 66 and 67; SEQ ID NO: 68(the variable region of an α chain of the anti-PDS5A_(Y1000F; H1007Y)TCR); SEQ ID NO: 69 (the variable region of a β chain of theanti-PDS5A_(Y1000F; H1007Y) TCR); both SEQ ID NOs: 68 and 69; SEQ ID NO:70 or 72 (the variable region of an α chain of an anti-MED13_(P1691S)TCR); SEQ ID NO: 71 or 73 (the variable region of a β chain of ananti-MED13_(P1691S) TCR); both SEQ ID NOs: 70 and 71; or both SEQ IDNOs: 72 and 73. Preferably, the nucleotide sequence comprises (a) SEQ IDNOs: 64-65; (b) SEQ ID NOs: 66-67; (c) SEQ ID NOs: 68-69; (d) SEQ IDNOs: 70-71; or (e) SEQ ID NOs: 72-73.

In an embodiment of the invention, the nucleic acid comprises acodon-optimized nucleotide sequence. Without being bound to a particulartheory or mechanism, it is believed that codon optimization of thenucleotide sequence increases the translation efficiency of the mRNAtranscripts. Codon optimization of the nucleotide sequence may involvesubstituting a native codon for another codon that encodes the sameamino acid, but can be translated by tRNA that is more readily availablewithin a cell, thus increasing translation efficiency. Optimization ofthe nucleotide sequence may also reduce secondary mRNA structures thatwould interfere with translation, thus increasing translationefficiency.

The invention also provides a nucleic acid comprising a nucleotidesequence which is complementary to the nucleotide sequence of any of thenucleic acids described herein or a nucleotide sequence which hybridizesunder stringent conditions to the nucleotide sequence of any of thenucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches from a random sequence that happened to have a fewsmall regions (e.g., 3-10 bases) that matched the nucleotide sequence.Such small regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive TCRs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. The recombinant expression vectors may be as describedherein with respect to other aspects of the invention.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described hereinand populations of host cells. The host cell, and populations thereof,may be as described herein with respect to other aspects of theinvention.

The inventive TCRs, polypeptides, proteins, nucleic acids, recombinantexpression vectors, and host cells (including populations thereof) canbe isolated and/or purified. The term “isolated” as used herein meanshaving been removed from its natural environment. The term “purified” asused herein means having been increased in purity, wherein “purity” is arelative term, and not to be necessarily construed as absolute purity.For example, the purity can be at least about 50%, can be greater than60%, 70%, 80%, 90%, 95%, or can be 100%.

Another embodiment of the invention provides an isolated population ofcells prepared according to any of the methods described herein withrespect to other aspects of the invention. The population of cells canbe a heterogeneous population comprising the host cells expressing theisolated TCR, or the antigen-binding portion thereof, in addition to atleast one other cell, e.g., a host cell (e.g., a PBMC), which does notexpress the isolated TCR, or the antigen-binding portion thereof, or acell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, anerythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, amuscle cell, a brain cell, etc. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly of host cells (e.g., consisting essentially of)expressing the isolated TCR, or the antigen-binding portion thereof. Thepopulation also can be a clonal population of cells, in which all cellsof the population are clones of a single host cell expressing theisolated TCR, or the antigen-binding portion thereof, such that allcells of the population express the isolated TCR, or the antigen-bindingportion thereof. In one embodiment of the invention, the population ofcells is a clonal population comprising host cells expressing theisolated TCR, or the antigen-binding portion thereof, as describedherein. By introducing the nucleotide sequence encoding the isolatedTCR, or the antigen binding portion thereof, into host cells, theinventive methods may, advantageously, provide a population of cellsthat comprises a high proportion of host cells that express the isolatedTCR and have antigenic specificity for the mutated amino acid sequence.In an embodiment of the invention, about 1% to about 100%, for example,about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100%, or arange defined by any two of the foregoing values, of the population ofcells comprises host cells that express the isolated TCR and haveantigenic specificity for the mutated amino acid sequence. Without beingbound to a particular theory or mechanism, it is believed thatpopulations of cells that comprise a high proportion of host cells thatexpress the isolated TCR and have antigenic specificity for the mutatedamino acid sequence have a lower proportion of irrelevant cells that mayhinder the function of the host cell, e.g., the ability of the host cellto target the destruction of cancer cells and/or treat or preventcancer.

The inventive TCRs, or the antigen-binding portions thereof,polypeptides, proteins, nucleic acids, recombinant expression vectors,host cells, and populations of cells (hereinafter, “inventive TCRmaterial(s)”) can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the inventive TCRs, or theantigen-binding portions thereof, polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, or populations of cells anda pharmaceutically acceptable carrier. The inventive pharmaceuticalcomposition can comprise an inventive TCR, or an antigen-binding portionthereof, or population of cells in combination with anotherpharmaceutically active agent(s) or drug(s), such as a chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used for the particular inventive TCR material underconsideration. Such pharmaceutically acceptable carriers are well-knownto those skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichhas no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive TCR material, as well as by the particular method used toadminister the inventive TCR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Suitable formulations may include any of those for oral,parenteral, subcutaneous, intravenous, intramuscular, intraarterial,intrathecal, or interperitoneal administration. More than one route canbe used to administer the inventive TCR material, and in certaininstances, a particular route can provide a more immediate and moreeffective response than another route.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive population of cells is to beadministered, the pharmaceutically acceptable carrier for the cells forinjection may include any isotonic carrier such as, for example, normalsaline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl inwater, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolytesolution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield,Ill.), about 5% dextrose in water, or Ringer's lactate. In anembodiment, the pharmaceutically acceptable carrier is supplemented withhuman serum albumin.

It is contemplated that the inventive TCR materials, and pharmaceuticalcompositions can be used in methods of treating or preventing cancer.Without being bound to a particular theory or mechanism, the inventiveTCRs, or the antigen-binding portions thereof, are believed to bindspecifically to a mutated amino acid sequence encoded by acancer-specific mutation, such that the TCR, or the antigen-bindingportion thereof, when expressed by a cell, is able to mediate an immuneresponse against a target cell expressing the mutated amino acidsequence. In this regard, the invention provides a method of treating orpreventing cancer in a patient, comprising administering to the patientany of the pharmaceutical compositions, TCRs, antigen-binding portionsthereof, polypeptides, proteins, nucleic acids, recombinant expressionvectors, host cells, or populations of cells described herein, in anamount effective to treat or prevent cancer in the patient.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a patient. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the cancer being treated orprevented. For example, treatment or prevention can include promotingthe regression of a tumor. Also, for purposes herein, “prevention” canencompass delaying the onset of the cancer, or a symptom or conditionthereof.

For purposes of the invention, the amount or dose of the inventive TCRmaterial or pharmaceutical composition administered (e.g., numbers ofcells when the inventive population of cells is administered) should besufficient to effect, e.g., a therapeutic or prophylactic response, inthe patient over a reasonable time frame. For example, the dose of theinventive TCR material or pharmaceutical composition should besufficient to bind to a mutated amino acid sequence encoded by acancer-specific mutation, or detect, treat or prevent cancer in a periodof from about 2 hours or longer, e.g., 12 to 24 or more hours, from thetime of administration. In certain embodiments, the time period could beeven longer. The dose will be determined by the efficacy of theparticular inventive TCR material or pharmaceutical compositionadministered and the condition of the patient, as well as the bodyweight of the patient to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which target cells are lysed or IFN-γ is secreted by T cellsexpressing the inventive TCR, or the antigen-binding portion thereof, orthe inventive populations of cells, upon administration of a given doseof such T cells to a mammal among a set of mammals of which is eachgiven a different dose of the cells, could be used to determine astarting dose to be administered to a patient. The extent to whichtarget cells are lysed or IFN-γ is secreted upon administration of acertain dose can be assayed by methods known in the art.

The dose of the inventive TCR material or pharmaceutical compositionalso will be determined by the existence, nature and extent of anyadverse side effects that might accompany the administration of aparticular inventive TCR material or pharmaceutical composition.Typically, the attending physician will decide the dosage of theinventive TCR material or pharmaceutical composition with which to treateach individual patient, taking into consideration a variety of factors,such as age, body weight, general health, diet, sex, inventive TCRmaterial or pharmaceutical composition to be administered, route ofadministration, and the severity of the condition being treated.

In an embodiment in which the inventive population of cells is to beadministered, the number of cells administered per infusion may vary,for example, in the range of one million to 200 billion cells; however,amounts below or above this exemplary range are within the scope of theinvention. For example, the daily dose of inventive host cells can beabout 1 million to about 200 billion cells (e.g., about 5 million cells,about 25 million cells, about 500 million cells, about 1 billion cells,about 5 billion cells, about 20 billion cells, about 30 billion cells,about 40 billion cells, about 60 billion cells, about 80 billion cells,about 100 billion cells, about 120 billion cells, about 130 billioncells, about 150 billion cells, about 160 billion cells, about 170billion cells, about 180 billion cells, about 190 billion cells, about200 billion cells, or a range defined by any two of the foregoingvalues), preferably about 10 million to about 200 billion cells (e.g.,about 20 million cells, about 30 million cells, about 40 million cells,about 60 million cells, about 70 million cells, about 80 million cells,about 90 million cells, about 10 billion cells, about 25 billion cells,about 50 billion cells, about 75 billion cells, about 90 billion cells,about 100 billion cells, about 110 billion cells, about 120 billioncells, about 130 billion cells, about 140 billion cells, about 150billion cells, about 160 billion cells, about 170 billion cells, about180 billion cells, about 190 billion cells, about 200 billion cells, ora range defined by any two of the foregoing values), more preferablyabout 100 million cells to about 200 billion cells (e.g., about 120million cells, about 250 million cells, about 350 million cells, about450 million cells, about 650 million cells, about 800 million cells,about 900 million cells, about 3 billion cells, about 30 billion cells,about 45 billion cells, about 50 billion cells, about 75 billion cells,about 90 billion cells, about 100 billion cells, about 110 billioncells, about 120 billion cells, about 130 billion cells, about 140billion cells, about 150 billion cells, about 160 billion cells, about170 billion cells, about 180 billion cells, about 190 billion cells,about 200 billion cells, or a range defined by any two of the foregoingvalues).

For purposes of the inventive methods, wherein populations of cells areadministered, the cells can be cells that are allogeneic or autologousto the patient. Preferably, the cells are autologous to the patient.

Another embodiment of the invention provides any of the TCR materials orpharmaceutical compositions described herein for use in treating orpreventing cancer in a patient.

The cancer may, advantageously, be any cancer, including any of acutelymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma,bone cancer, brain cancer, breast cancer, cancer of the anus, analcanal, or anorectum, cancer of the eye, cancer of the intrahepatic bileduct, cancer of the joints, cancer of the neck, gallbladder, or pleura,cancer of the nose, nasal cavity, or middle ear, cancer of the oralcavity, cancer of the vagina, cancer of the vulva, cholangiocarcinoma,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, uterine cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, urinary bladdercancer, solid tumors, and liquid tumors. Preferably, the cancer is anepithelial cancer. In an embodiment, the cancer is cholangiocarcinoma,melanoma, colon cancer, or rectal cancer.

The mammal referred to in the inventive methods can be any mammal. Asused herein, the term “mammal” refers to any mammal, including, but notlimited to, mammals of the order Rodentia, such as mice and hamsters,and mammals of the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). Preferably, the mammals are from the orderArtiodactyla, including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). Preferably, the mammals areof the order Primates, Ceboids, or Simoids (monkeys) or of the orderAnthropoids (humans and apes). A more preferred mammal is the human. Inan especially preferred embodiment, the mammal is the patient expressingthe cancer-specific mutation.

In an embodiment of the invention, TCR(s), or antigen-binding portion(s)thereof, may be isolated from the T cells that express PD-1 immediatelyafter separating the T cells that express PD-1 from cells that do notexpress PD-1. These TCR(s), or antigen-binding portion(s) thereof, maybe cloned into a recombinant expression vector, and introduced into hostcells to obtain expression of the TCR(s), or antigen binding portion(s)thereof, by the host cells. The host cells that express the TCR(s), orantigen binding portions thereof, could then be screened for antigenicspecificity for a mutated amino acid sequence encoded by acancer-specific mutation.

In this regard, an embodiment of the invention provides a method ofisolating T cells having antigenic specificity for a mutated amino acidsequence encoded by a cancer-specific mutation, the method comprisingobtaining a first population of PBMCs from a sample of peripheral bloodfrom a patient; selecting T cells that express PD-1 from the bulkpopulation; separating the T cells that express PD-1 from cells that donot express PD-1 to obtain a T cell population enriched for T cells thatexpress PD-1; isolating nucleotide sequence(s) that encode(s) one ormore TCR(s), or antigen-binding portion(s) thereof, from the T cells ofthe population enriched for T cells that express PD-1; introducing thenucleotide sequence(s) encoding the TCR(s), or antigen bindingportion(s) thereof, into further population(s) of PBMCs to obtain Tcells that express the TCR(s), or antigen binding portion(s) thereof;identifying one or more genes in the nucleic acid of a cancer cell ofthe patient, each gene containing a cancer-specific mutation thatencodes a mutated amino acid sequence; inducing autologous APCs of thepatient to present the mutated amino acid sequence; co-culturing the Tcells that express the TCR(s), or antigen binding portion(s) thereof,with the autologous APCs that present the mutated amino acid sequence;and selecting the T cells that (a) were co-cultured with the autologousAPCs that present the mutated amino acid sequence and (b) have antigenicspecificity for the mutated amino acid sequence presented in the contextof a MHC molecule expressed by the patient.

Obtaining a first population of PBMCs from a sample of peripheral blood;selecting T cells that express PD-1; and separating the T cells thatexpress PD-1 from cells that do not express PD-1 may be carried out asdescribed herein with respect to other aspects of the invention.

The method may further comprise isolating nucleotide sequence(s) thatencode(s) one or more TCR(s), or antigen binding portion(s) thereof,from the T cells of the population enriched for T cells that expressPD-1. While the method may further comprise expanding the numbers of theT cells that express PD-1 prior to isolating the nucleotide sequence, ina preferred embodiment, the method comprises isolating the nucleotidesequence from the T cells without expanding the numbers of the T cellsthat express PD-1 prior to isolating the nucleotide sequence. Forexample, the TCR, or antigen binding portion thereof, may be isolatedfrom a single cell. In an embodiment of the invention, the methodcomprises isolating nucleotide sequence(s) that encode(s) at least oneTCR, or antigen binding portion thereof. However, the number of TCR(s),or antigen binding portion(s) thereof, that may be isolated using theinventive methods is not limited and may include more than one TCR(s),or antigen binding portion(s) thereof (for example, about 2, about 3,about 4, about 5, about 10, about 11, about 12, about 13, about 14,about 15, about 20, about 25, about 30, about 40, about 50, about 60,about 70, about 80, about 90, about 100, about 150, about 200, about400, about 600, about 800, about 1000, about 1500, about 2000 or more,or a range defined by any two of the foregoing values). The nucleotidesequence(s) that encode(s) one or more TCR(s), or antigen bindingportion(s) thereof, may otherwise be isolated as described herein withrespect to other aspects of the invention.

The method may further comprise introducing the nucleotide sequence(s)encoding the TCR(s), or antigen binding portion(s) thereof, into furtherpopulation(s) of PBMCs to obtain T cells that express the TCR(s), orantigen binding portion(s) thereof. Each TCR, or antigen binding portionthereof, isolated according to this embodiment of the invention may beintroduced into a different population of PBMCs to provide multiplepopulations of cells, each population of cells expressing a differentTCR or antigen binding portion thereof. Introducing the nucleotidesequence(s) encoding the TCR(s), or antigen binding portion(s) thereof,into further population(s) of PBMCs may otherwise be carried out asdescribed herein with respect to other aspects of the invention.

Identifying one or more genes in the nucleic acid of a cancer cell ofthe patient; inducing APCs of the patient to present the mutated aminoacid sequence; co-culturing the T cells with the autologous APCs thatpresent the mutated amino acid sequence; and selecting the T cells that(a) were co-cultured with the autologous APCs that present the mutatedamino acid sequence and (b) have antigenic specificity for the mutatedamino acid sequence may all be carried out as described herein withrespect to other aspects of the invention. In an embodiment of theinvention in which more than one TCR, or antigen binding portionthereof, is isolated and a nucleotide sequence encoding each TCR, orantigen binding portion thereof is introduced into a differentpopulation of cells, co-culturing may comprise separately co-culturingeach population of cells (each expressing a different TCR, or antigenbinding portion thereof) with the autologous APCs. Selecting maycomprise determining which TCR, or antigen binding portion thereof, hasantigenic specificity for the mutated amino acid sequence (e.g., byprocess of elimination). In an embodiment of the invention, the numbersof selected cells may be expanded as described herein with respect toother aspects of the invention. In an embodiment of the invention, thenumbers of selected cells are not expanded.

In an embodiment of the invention, the method may further compriseisolating a nucleotide sequence that encodes a TCR, or anantigen-binding portion thereof, from the selected T cells that haveantigenic specificity for the mutated amino acid sequence, wherein theTCR, or the antigen-binding portion thereof, has antigenic specificityfor the mutated amino acid sequence. Isolating a nucleotide sequencethat encodes a TCR, or an antigen-binding portion thereof, from theselected T cells may be carried out as described herein with respect toother aspects of the invention. Further embodiments of the invention mayprovide methods of preparing a population of cells that expresses theTCR, or antigen binding portion thereof; a TCR, or an antigen-bindingportion thereof, isolated according to the inventive methods; isolatedpopulations of cells prepared according to the inventive methods;pharmaceutical compositions comprising the inventive TCR, or antigenbinding portion thereof, or the inventive population of cells; andmethods of treating cancer using the inventive compositions, all ofwhich may be as described herein with respect to other aspects of theinvention.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the expression of PD-1 and TIM-3 in the CD8+cell population in the peripheral blood of a melanoma patient and thepurity of the cells separated according to PD-1 and TIM-3.

PBMC from melanoma patient 3713 were rested overnight in the absence ofIL-2, stained with antibodies, and sorted according to expression of CD8and CD3 by FACS. Then, the CD3+CD8+ cells were sorted according toexpression of PD-1 and TIM-3 by FACS. The gates of the stained sampleswere set based on the isotype control. The frequency of the CD8+ PBMCpopulations expressing each of the markers is indicated in Table 1below.

TABLE 1 Percentage of cells expressing Population Phenotype indicatedphenotype Non-specific TIM-3+PD-1+ 0.1 Staining TIM-3−PD-1+ 4.4TIM-3+PD-1− 1.0 TIM-3−PD-1− 94.5 PD-1− TIM-3+PD-1+ 0.0 TIM-3−PD-1+ 0.0TIM-3+PD-1− 2.0 TIM-3−PD-1− 98.0 PD-1+ TIM-3+PD-1+ 14.3 TIM-3−PD-1+ 77.6TIM-3+PD-1− 2.0 TIM-3−PD-1− 6.1 PD-1hi TIM-3+PD-1+ 3.3 TIM-3−PD-1+ 93.3TIM-3+PD-1− 0.0 TIM-3−PD-1− 3.3 TIM-3+ TIM-3+PD-1+ 1.9 TIM-3−PD-1+ 0.0TIM-3+PD-1− 83.0 TIM-3−PD-1− 15.1 PD-1+TIM-3+ TIM-3+PD-1+ 83.3TIM-3−PD-1+ 16.7 TIM-3+PD-1− 0.0 TIM-3−PD-1− 0.0

Example 2

This example demonstrates that CD8+PD-1+, CD8+PD-1+TIM-3−, andCD8+PD-1+TIM-3+ cell populations, but not bulk CD8+, CD8+PD-1−,CD8+TIM-3−, or CD8+TIM-3+ cell populations, isolated from peripheralblood recognize target cells pulsed with unique, patient-specificmutated epitopes.

Pheresis from a melanoma patient (3713) was thawed and rested overnightin the absence of cytokines. CD8+ cells were sorted according to PD-1and TIM-3 expression into the following populations: CD8+ bulk,CD8+PD-1−, CD8+PD-1+, CD8+TIM-3−, CD8+TIM-3+, CD8+PD-1+TIM-3−, andCD8+PD-1+TIM-3+. The numbers of the sorted cells were expanded in vitrofor 15 days. On day 15, the cells were washed and co-cultured withtarget autologous B cells pulsed with wild type (wt) or mutated (mut)epitopes known to be recognized by the patient's tumor-infiltratinglymphocytes at a ratio of 2×10⁴ effector cells: 1×10⁵B cells. T cellswere also co-cultured with the autologous tumor cell line (TC3713) inthe absence or presence of HLA-I blocking antibody W6/32 or with anallogeneic tumor cell line (TC3903). T cells were also co-cultured withanti-CD3 antibody as a control. Reactivity was assessed by quantifyingIFN-gamma spots 16 hours (h) after the co-culture by IFN-γ ELISpot. Theresults are shown in Tables 2A and 2B.

As shown in Tables 2A and 2B, CD8+PD-1+, CD8+PD-1+TIM-3−, andCD8+PD-1+TIM-3+ cell populations, but not bulk CD8+, CD8+PD-1−,CD8+TIM-3−, or CD8+TIM-3+ cell populations, isolated from peripheralblood recognized target cells pulsed with unique, patient-specificmutated epitopes.

TABLE 2A Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset Epitope CD8+ CD8+PD-1− CD8+PD-1+ Notarget 1 1 2 CEF peptide pool 72 58 4 WDR wt 1 4 0 WDR mut 5 0 >750 SRPXwt 2 2 1 SRPX mut 11 1 77 AFMID wt 3 1 61 AFMID mut 3 1 246 HELZ2 wt 3 29 HELZ2 mut 0 1 219 PLSCR4 wt 1 2 0 PLSCR4 mut 5 1 2 GCN1L1 wt 2 1 2GCN1L1 mut 2 0 5 CENPL wt 1 0 0 CENPL mut 3 1 >750 AHNAK wt 1 0 2 AHNAKmut 1 0 5 TC3713 17 24 >750 TC3713 + W6/32 0 0 44 TC3903 8 11 9Anti-CD3 >750 >750 >750

TABLE 2B Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset CD8+TIM- CD8+TIM- CD8+PD- CD8+PD- Epitope3− 3+ 1+TIM-3− 1+TIM-3+ No target 0 0 0 0 CEF peptide 40 8 25 1 pool WDRwt 1 3 5 0 WDR mut 1 0 257 2 SRPX wt 4 1 14 2 SRPX mut 3 2 381 104 AFMIDwt 2 0 59 2 AFMID mut 2 1 88 1 HELZ2 wt 4 0 21 20 HELZ2 mut 3 0 465 341PLSCR4 wt 0 0 11 0 PLSCR4 mut 2 0 5 0 GCN1L1 wt 2 1 6 0 GCN1L1 mut 1 110 2 CENPL wt 2 1 8 0 CENPL mut 2 1 53 1 AHNAK wt 4 0 4 0 AHNAK mut 0 2212 1 TC3713 7 76 >750 >750 TC3713 + 0 2 85 17 W6/32 TC3903 18 12 22 1Anti-CD3 >750 >750 >750 >750

Example 3

This example demonstrates that CD8+PD-1+, CD8+PD-1+TIM-3−,CD8+PD-1+TIM-3+, and CD8+PD-1+CD27hi cell populations, but not bulkCD8+, CD8+TIM-3−, CD8+TIM-3+, CD8+PD-1-CD27hi, or CD8+PD-1− cellpopulations, isolated from peripheral blood recognize target cellselectroporated with RNA encoding unique, patient-specific mutatedepitopes.

Pheresis from melanoma patient 3903 was thawed and rested overnight inthe absence of cytokines. CD8+ cells were enriched by bead separationand then sorted according to PD-1 and TIM-3 expression into thefollowing populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, CD8+TIM-3−,CD8+TIM-3+, CD8+PD-1+TIM-3−, CD8+PD-1+TIM-3+, CD8+PD-1-CD27hi, andCD8+PD-1+CD27hi. The numbers of sorted cells were expanded in vitro for15 days. On day 15, the cells were washed and co-cultured with targetautologous dendritic cells electroporated with RNA encoding mutatedtandem minigenes (TMGs 1-26; each encoding multiple 25mers containing amutation flanked by the endogenous sequence) identified by exomesequencing of a tumor from patient 3903. The effector cells wereco-cultured with the target cells at a ratio of 2×10⁴ effector cells: toabout 1×10⁵ DCs. The effector cells were also co-cultured with theautologous tumor cell line (TC3903) or with an allogeneic tumor cellline (TC3903). Reactivity was assessed by quantifying IFN-gamma spots 16h after the co-culture by IFN-γ ELISpot. The results are shown in Tables3A-3C.

As shown in Tables 3A-3C, CD8+PD-1+, CD8+PD-1+TIM-3−, CD8+PD-1+TIM-3+,and CD8+PD-1+CD27hi cell populations, but not bulk CD8+, CD8+TIM-3−,CD8+TIM-3+, CD8+PD-1-CD27hi, or CD8+PD-1− cell populations, isolatedfrom peripheral blood recognized target cells electroporated with RNAencoding unique, patient-specific mutated epitopes. In melanoma patient3903, CD8+ PBL subsets expressing PD-1 were enriched in cellsrecognizing TMG-9 (Tables 2A-2B). In this patient, further enrichment inmutation-specific cells from peripheral blood was observed whenselecting CD8+ cells expressing PD-1 in combination with TIM-3 or CD27(TMG-8, TMG-3, and weaker recognition of TMG-7 and TMG-11) (Tables3A-3C).

CD8+ lymphocytes expressing PD-1 in the peripheral blood of patient 3903were enriched in cells capable of recognizing the autologous tumor cellline (Tables 3A-3C).

The sorted cells were also co-cultured with autologous DCselectroporated with RNA encoding full-length MART-1, GP100, tyrosinase,NY-ESO-1, MAGE-A3, or SSX2. CD8+ lymphocytes expressing PD-1 in theperipheral blood of patient 3903 also recognized mutation-specific cellsand cancer germline antigens SSX2 and MAGE-A3.

TABLE 3A Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset Epitope CD8+ CD8+PD-1− CD8+PD-1+ Notarget 0 0 0 CEF peptide 44 119 >500 pool irrelevant 0 1 1 TMG TMG-1 0 00 TMG-2 0 3 1 TMG-3 4 5 0 TMG-4 9 1 3 TMG-5 9 1 1 TMG-6 22 2 6 TMG-7 2 10 TMG-8 7 4 15 TMG-9 9 0 303 TMG-10 1 1 0 TMG-11 8 2 29 TMG-12 11 5 11TMG-13 1 2 2 TMG-14 9 21 1 TMG-15 3 12 40 TMG-16 0 0 0 TMG-17 2 3 2TMG-18 4 1 0 TMG-19 0 3 0 TMG-20 1 1 1 TMG-21 0 2 1 TMG-22 2 3 3 TMG-230 0 1 TMG-24 0 1 12 TMG-25 0 4 17 TMG-26 1 0 3 DMSO 0 0 0 Peptide 1 1 9Nos 8-2 Peptide 3 0 >500 nos. 9-4 TC3903 2 0 >500 TC3713 41 17 12Anti-CD3 >500 >500 >500 1 μg/ml

TABLE 3B Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset CD8+TIM- CD8+TIM- CD8+PD- CD8+PD- Epitope3− 3+ 1+TIM-3− 1+TIM-3+ No target 0 0 0 0 CEF peptide 35 7 280 0 poolirrelevant 3 23 0 4 TMG TMG-1 3 3 1 3 TMG-2 5. 13 3 3 TMG-3 1 17 3 6TMG-4 3 15 0 3 TMG-5 2 17 7 50 TMG-6 0 9 2 0 TMG-7 4 14 8 224 TMG-8 3 079 >500 TMG-9 9 11 639 426 TMG-10 3 1 1 4 TMG-11 18 0 38 204 TMG-12 1151 24 63 TMG-13 2 21 3 1 TMG-14 27 57 11 0 TMG-15 5 35 4 16 TMG-16 3 152 1 TMG-17 2 28 2 8 TMG-18 1 26 5 19 TMG-19 0 8 0 0 TMG-20 1 16 1 0TMG-21 1 9 1 0 TMG-22 3 22 4 4 TMG-23 1 4 4 2 TMG-24 3 13 34 9 TMG-25 822 3 2 TMG-26 0 7 0 0 DMSO 0 0 2 0 Peptide 2 27 70 >500 Nos 8-2 Peptide0 2 >500 10 nos. 9-4 TC3903 1 34 227 365 TC3713 13 1 3. 109Anti-CD3 >500 >500 >500 >500 1 μg/ml

TABLE 3C Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset Epitopes CD8+PD-1−CD27hi CD8+PD-1+CD27hiNo target 0 0 CEF peptide 79 422 pool irrelevant 6 2 TMG TMG-1 2 0 TMG-22 3 TMG-3 8 306 TMG-4 4 0 TMG-5 3 8 TMG-6 2 0 TMG-7 3 0 TMG-8 7 82 TMG-94 395 TMG-10 0 21 TMG-11 1 52 TMG-12 5 12 TMG-13 1 5 TMG-14 5 4 TMG-15 97 TMG-16 3 0 TMG-17 1 2 TMG-18 0 22 TMG-19 0 0 TMG-20 1 0 TMG-21 3 1TMG-22 1 1 TMG-23 0 0 TMG-24 2 0 TMG-25 2 0 TMG-26 2 0 DMSO 1 0 Peptide0 58 Nos 8-2 Peptide 0 401 nos. 9-4 TC3903 0 >500 TC3713 1 13 Anti-CD3222 303 1 μg/ml

Example 4

This example demonstrates that CD8+PD-1+, CD8+PD-1hi, CD8+PD-1+TIM-3+,CD8+PD-1+CD27hi, and CD8+PD-1+CD27− cell populations, but not bulk CD8+,CD8+TIM-3−, CD8+TIM-3+, or CD8+PD-1− cell populations, isolated fromperipheral blood recognize target cells electroporated with RNA encodingunique, patient-specific mutated epitopes.

Pheresis from melanoma patient 3784 was thawed and rested overnight inthe absence of cytokines. CD8+ cells were enriched by bead separationand then sorted according to PD-1 and TIM-3 expression into thefollowing populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, CD8+PD-1hi,CD8+TIM-3−, CD8+TIM-3+, CD8+PD-1+TIM-3+, CD8+PD-1+CD27hi, andCD8+PD-1+CD27−. The numbers of sorted cells were expanded in vitro for15 days. On day 15, the cells were washed and co-cultured with targetautologous dendritic cells electroporated with RNA encoding mutatedtandem minigenes (TMGs 1-9; each encoding multiple 25mers containing amutation flanked by the endogenous sequence) identified by exomesequencing of a tumor from patient 3784. The effector cells wereco-cultured with the target cells at a ratio of 2×10⁴ effector cells: toabout 1×10⁵ DCs. The effector cells were also co-cultured withautologous DCs electroporated with RNA encoding epitopes forcytomegalovirus (CMV), Epstein-Barr virus (EBV), FLU (CEF), or anirrelevant TMG. T cells were also co-cultured with the autologous tumorcell line (TC3784) or with an allogeneic tumor cell line (TC3903).Reactivity was assessed by quantifying IFN-gamma spots 16 h after theco-culture by IFN-γ ELISpot.

The results are shown in Tables 4A-4C. As shown in Tables 4A-4C,CD8+PD-1+, CD8+PD-1hi, CD8+PD-1+TIM-3+, CD8+PD-1+CD27hi, andCD8+PD-1+CD27− cell populations, but not bulk CD8+, CD8+TIM-3−,CD8+TIM-3+, or CD8+PD-1− cell populations, isolated from peripheralblood recognized target cells electroporated with RNA encoding unique,patient-specific mutated epitopes.

In this patient, the peripheral blood CD8+ lymphocytes expressing PD-1were enriched in mutation-specific cells recognizing up to threeantigens (TMG-3, TMG-5, and TMG-8). Peripheral blood CD8+PD-1+ andPD-1hi T cells also recognized gp100.

Further separation of peripheral blood CD8+PD-1+ lymphocytes into CD27hior CD27− separated the lymphocytes recognizing TMG-3 from thoserecognizing TMG-5 and TMG-8.

The co-culture of the sorted cells with the autologous tumor cell lineor the allogeneic tumor cell line revealed that peripheral blood CD8+lymphocytes expressing PD-1 alone or in combination with TIM-3 or CD27were enriched in tumor-reactive cells.

TABLE 4A Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset Epitope CD8+ CD8+PD-1− CD8+PD-1+CD8+PD-1hi No target 0 0 0 0 CEF peptide 259 138 10. 57 pool irrelevant18 3 11 14 TMG TMG-1 11 0 4 6 TMG-2 7 0 6 4 TMG-3 14 0 77 291 TMG-4 7 09 39 TMG-5 7 2 88 77 TMG-6 24 2 3 10 TMG-7 11 1 9 2 TMG-8 18 0 217 111TMG-9 13 0 6 8 MART-1 5 1 48 7 GP100 27 1 154 418 TYR 17 2 9 6 MAGE-A314 2 29 156 NY-ESO-1 9 0 6 33 SSX2 16 0 0 33 TC3784 120 41 491 >500TC3903 22 18 129 212 Anti-CD3 >500 424 >500 >500

TABLE 4B Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset Epitope CD8+TIM-3− CD8+TIM-3+CD8+PD-1+TIM-3+ CD8+PD-1+CD27hi CD8+PD-1+CD27− No target 2 0 0 0 1 CEFpeptide 152 27 3 4 45 pool irrelevant 1 2 6 6 10 TMG TMG-1 0 2 0 0 11TMG-2 0 1 2 2 4 TMG-3 1 0 98 12 276 TMG-4 1 1 2 1 3 TMG-5 0 2 87 319 3TMG-6 0 1 3 1 4 TMG-7 0 0 6 0 3 TMG-8 5 1 402 175 5 TMG-9 0 0 2 4 2MART-1 3 24 3 0 222 GP100 11 36 >500 362 381 TYR 2 0 7 3 6 MAGE-A3 1 0304 91 6 NY-ESO-1 3 3 15 6 23 SSX2 1 1 16 4 5 TC3784 100 292 >500 500223 TC3903 30 23 26 177 76 Anti-CD3 482 489 >500 492 >500

Example 5

This example demonstrates that CD8+PD-1+ and CD8+PD-1hi cellpopulations, but not bulk CD8+ or CD8+PD-1− cell populations, isolatedfrom peripheral blood recognize target cells electroporated with RNAencoding unique, patient-specific mutated epitopes.

Pheresis from a colorectal cancer patient 3971 was thawed and restedovernight in the absence of cytokines. CD8+ cells were enriched by beadseparation and then sorted according to PD-1 expression into thefollowing populations: CD8+ bulk, CD8+PD-1−, CD8+PD-1+, and CD8+PD-1hi.The numbers of sorted cells were expanded in vitro for 15 days. On day15, the cells were washed and co-cultured with target autologousdendritic cells electroporated with RNA encoding mutated tandemminigenes (TMGs 1-9; each encoding multiple 25mers containing a mutationflanked by the endogenous sequence) identified by exome sequencing fromthe patient's tumor (at a ratio of 2×10⁴ effector cells: about 1×10⁵DCs). TMG-1 encoded mutated CASP8 peptide. The cells were alsoco-cultured with cells electroporated with RNA encoding a mock (empty)control vector or irrelevant TMG. Reactivity was assessed by quantifyingIFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot. The resultsare shown in Table 5. As shown in Table 5, CD8+PD-1+ and CD8+PD-4hi cellpopulations, but not bulk CD8+ or CD8+PD-1− cell populations, isolatedfrom peripheral blood recognized target cells electroporated with TMG-1or TMG-3 RNA.

TABLE 5 Number of IFN-γ spots measured per 2 × 10⁴ effector cells ineach blood-derived CD8+ subset Epitope CD8+ CD8+PD-1− CD8+PD-1+CD8+PD-1hi No target 0 0 0 0 irrelevant 1 1 0 0 TMG TMG-1 2 7 41 277TMG-2 0 0 0 0 TMG-3 2 0 3 175 TMG-4 1 2 1 0 TMG-5 0 0 0 0 TMG-6 2 3 7 0TMG-7 0 1 1 0 TMG-8 0 1 0 0 TMG-9 0 0 1 1 Anti-CD3 >500 >500 >500 >500

Example 6

This example demonstrates the isolation of a nucleotide sequenceencoding a TCR having antigenic specificity for target cellselectroporated with RNA encoding unique, patient-specific mutatedepitopes from a CD8+PD-1hi cell population.

The TMG-1 and TMG-3 reactive cells present in the CD8+PD-1hi cellpopulation of Example 5 (colorectal cancer patient) were selected byFACS based on the upregulation of 4-1BB (CD137). On day 15, PD-1hi bulkcells, as well as CD137−, and CD137+ fractions, were co-cultured withtarget DCs electroporated with RNA encoding for TMG-1 or TMG-3, orplate-bound OKT3. Reactivity was assessed by CD137 upregulation after 20h. The number of cells and the percentage of cells (with respect to bulkcells) having the indicated phenotype are shown in Table 6A.

TABLE 6A Target cells co-cultured Gated on live cells, single cells,CD3+CD8+ cells with CD8+PD- Irrelevant 1hi cells TMG TMG-1 TMG-3 CD137−99.8 99.1 99.2 (1.7 × 10⁵ cells) (1.7 × 10⁵ cells) (1.7 × 10⁵ cells)CD137+ 0.0 0.3 0.2 (634 cells) (489 cells)

The numbers of cells in Table 6A were expanded in vitro for 14 days. Thecell yields obtained are shown in Table 6B.

TABLE 6B Target cells co-cultured with CD8+PD- Irrelevant 1hi cells TMGTMG-1 TMG-3 CD137− 1.4 × 10⁸ 1.8 × 10⁸ 1.4 × 10⁸ CD137+ — 8.5 × 10⁷ 7.8× 10⁷

A nucleotide sequence encoding a TCR was isolated from the TMG-1 andTMG-3 reactive cells that were selected on the basis of CD137upregulation. The CD137+ TMG-1 reactive cells (>97% one clonotype)comprised an alpha chain CDR3 amino acid sequence of CAVRDRGTGGFKTIF(SEQ ID NO: 1) and a beta chain CDR3 amino acid sequence ofCASITKDRAYEQYF (SEQ ID NO: 2). The CD137+ TMG-3 reactive cells (>93% oneclonotype) comprised an alpha chain CDR3 amino acid sequence ofCAYRSASDMRF (SEQ ID NO: 3) and a beta chain CDR3 amino acid sequence ofCASSPETGGISEQYF (SEQ ID NO: 4).

Accordingly, the selection of CD137+ cells that were reactive againsttarget cells electroporated with TMG-1 or TMG-3 from CD8+PD-1hilymphocytes sorted from the peripheral blood led to the generation ofhighly enriched TMG-1 and TMG-3 specific populations, each encoding forone dominant TCR.

Example 7

This example demonstrates the identification of the mutation recognizedby TMG-1 reactive cells isolated from CD8+PD-1+ peripheral blood cells.

Following 15 days in culture, the sorted TMG-1-reactive, CD137− andCD137+ effector populations of Example 6 were co-cultured withautologous DCs that were electroporated with TMG-1 RNA or were pulsedwith wild type or mutated CASP8 minimal epitopes. Reactivity wasassessed by quantifying IFN-gamma spots 16 h after the co-culture byIFN-γ ELISpot. The resulting numbers of IFN-γ spots measured per 2×10⁴cells are shown in Table 7.

TABLE 7 Sorted vs TMG-1 PD-1hi bulk CD137− CD137+ No target 0 0.0 0irrelevant TMG 0 0 0 TMG-1 120 11 >500 wt CASP8 0 0 1 mut CASP8 7014 >500 anti-CD3 >500 >500 >500

As shown in Table 7, the TMG-1 reactive cells enriched from peripheralblood recognized a unique mutation in CASP8 identified through exomesequencing of 3971 tumor.

Example 8

This example demonstrates the identification of the mutation recognizedby TMG-3 reactive cells isolated from CD8+PD-1+ peripheral blood cells.

Following 15 days in culture, the sorted TMG-3-reactive, CD137− andCD137+ effector cell populations of Example 6 were co-cultured withautologous DCs that were pulsed with mutated long peptides (μg/ml)derived from TMG-3 (Nos. 1-16 in Table 8). Reactivity was assessed byquantifying IFN-gamma spots 16 h after the co-culture by IFN-γ ELISpot.The resulting numbers of IFN-γ spots measured per 2×10⁴ cells are shownin Table 8.

TABLE 8 Sorted vs. TMG-3 Long peptide# CD8+PD-1hi bulk CD137− CD137+DMSO 0 0 0 1 61 21 >500 2 0 1 0 3 1 0 2 4 0 0 4 5 2 0 1 6 0 0 0 7 1 0 48 0 0 2 9 0 0 1 10 0 0 1 11 0 0 0 12 0 0 0 13 0 0 0 14 0 0 0 15 0 0 1 160 0 1 Anti-CD3 1 μg/ml >500 >500 >500

As shown in Table 8, the TMG-3 reactive cells enriched from CD8+PD-hipopulation selected from peripheral blood recognized long peptide TMG-3number 1, which encoded a mutated HISTH3B peptide.

Example 9

This example demonstrates the reactivity of PBL engineered to expressthe mutated CASP8 peptide specific T-cell receptor isolated in Example6.

PBL were transduced with the nucleotide sequence encoding the TMG-1specific TCR of Example 6 or an empty vector (control). Autologous Bcells were pulsed for 2 h with either wild type or mutated CASP8peptides. The pulsed cells were co-cultured with TCR transduced orvector transduced cells (at a ratio of 2×10⁵ B cells: 2×10⁴ effectorcells). Reactivity was measured by 4-1BB upregulation 24 h later. Thefrequency of 4-1BB within the CD3+CD8+ cells is shown in FIG. 1. Asshown in FIG. 1, PBL engineered to express the CASP8 mut specific T-cellreceptor isolated in Example 6 were reactive against the mutated CASP8peptide.

Example 10

This example demonstrates that CD8+PD-1+ and CD8+PD-1hi cellpopulations, but not bulk CD8+ or CD8+PD-1− cell populations, isolatedfrom peripheral blood recognize target cells pulsed with unique,patient-specific mutated epitopes. This example also demonstrates thatCD4+PD-1+ and CD4+PD-1hi cell populations, but not bulk CD4+ orCD4+PD-1− cell populations, isolated from peripheral blood recognizetarget cells electroporated with RNA encoding NY-ESO-1.

Pheresis from a melanoma patient (3998) was thawed and rested overnightin the absence of cytokines. CD8+ cells were sorted according to PD-1expression into the following populations: CD8+ bulk, CD8+PD-1−,CD8+PD-1+, and CD8+PD-1hi as described in Example 2. CD4+ cells weresorted according to PD-1 expression into the following populations: CD4+bulk, CD4+PD-1-, CD4+PD-1+, and CD4+PD-1hi as described in Example 2.The numbers of the sorted cells were expanded in vitro for 15 days. Onday 15, the cells were washed and co-cultured with target autologous DCselectroporated with RNA encoding mutated tandem minigenes (TMGs 1-7;each encoding multiple 25mers containing a mutation flanked by theendogenous sequence) identified by exome sequencing of a tumor frompatient 3998, or RNA encoding MART-1, gp100, tyrosinase, NY-ESO-1,MAGE-A3, or SSX2. The cells were also co-cultured with autologous tumorcell line or allogeneic tumor cell line (3713). T cells were alsoco-cultured with anti-CD3 antibody as a control. Reactivity was assessedby quantifying IFN-gamma spots 16 hours (h) after the co-culture byIFN-γ ELISpot. The results are shown in Tables 9A and 9B.

As shown in Table 9A, the CD8+PD-1+ and CD8+PD-1hi cell populations, butnot bulk CD8+ or CD8+PD-1− cell populations, isolated from peripheralblood recognized target cells electroporated with RNA encoding withunique, patient-specific mutated epitopes (TMG-1). As shown in Table 9A,the CD8+PD-1hi cell population, but not bulk CD8+, CD8+PD-1+, orCD8+PD-1− cell populations, isolated from peripheral blood recognizedtarget cells electroporated with RNA encoding with unique,patient-specific mutated epitopes (TMG-3). The CD8+PD-1+ and CD8+PD-1hicell populations isolated from peripheral blood recognized target cellselectroporated with RNA encoding NY-ESO-1 (Table 9A).

TABLE 9A Cells isolated from Pheresis of 3998 CD8+ CD8+PD-1− CD8+PD-1+CD8+PD-1hi No target 0 1 0 0 Irrel. TMG 2 15 1 0 CEF 84 43 53 39 TMG-167 34 394 478 TMG-2 11 15 6 4 TMG-3 16 7 56 159 TMG-4 4 24 7 0 TMG-5 4 436 11 TMG-6 0 5 2 0 TMG-7 9 19 2 1 MART-1 3 11 2 2 GP-100 11 28 16 5Tyrosinase 11 15 3 8 NY-ESO-1 179 34 >500 >500 MAGE-A3 6 6 6 0 SSX2 1213 9 24 TC3998 110 63 >500 >500 TC3713 215 229 150 10Anti-CD3 >500 >500 >500 >500 1 μg/ml

TABLE 9B Cells isolated from Pheresis of 3998 CD4+ CD4+PD-1− CD4+PD-1+CD4+PD-1hi No target 3 0 0 3 Irrel. TMG 1 30 35 6 CEF 8 6 36 6 TMG-1 2620 12 11 TMG-2 14 20 14 14 TMG-3 26 15 21 20 TMG-4 30 29 19 6 TMG-5 5 617 8 TMG-6 3 9 6 8 TMG-7 24 24 12 4 MART-1 56 17 13 6 GP-100 14 25 24 18Tyrosinase 29 24 36 7 NY-ESO-1 42 29 320 >500 MAGE-A3 13 19 13 17 SSX224 20 14 10 TC3998 22 16 170. 84 TC3713 34 57 16 15Anti-CD3 >500 >500 >500 >500 1 μg/ml

As shown in Table 9B, CD4+PD-1+ and CD4+PD-1hi cell populations, but notbulk CD4+ or CD4+PD-1− cell populations, isolated from peripheral bloodrecognized target cells electroporated with RNA encoding NY-ESO-1.

Examples 11-17

The following materials and methods were employed for the experimentsdescribed in Examples 11-17.

Subjects, Tumor Biopsies, and PBMCs.

Leukapheresis products, and tumor samples were obtained from individualswith stage IV melanoma enrolled on a clinical protocol (03-C-0277),approved by the institutional-review board (IRB) of the National CancerInstitute (NCI). Informed consent was obtained from all subjects, andthey all had progressive disease at the time of sample acquisition. The5 individuals studied in detail were chosen on the basis of availabilityof pre-treatment leukapheresis, and matched frozen fresh tumor toperform whole-exome sequencing and transcriptome analysis. Patients wereeither treatment naïve (NCI-3998), or had undergone prior therapiesincluding surgery, chemotherapy, and immunotherapy (NCI-3713, 3784,3903, and 3926). The patient characteristics are provided in Table 10.The patients that received prior therapies had been last treated from7-55 months before the leukapheresis product was obtained. A summary ofthe individuals included in the phenotypic characterization ofcirculating and tumor-infiltrating lymphocytes is provided in Table 11.Melanoma specimens were surgically resected and digested into singlecell suspensions using the GENTLEMACS Dissociator (Miltenyi Biotec,Gladbach, Germany) as described in Gros et al., J. Clin. Invest., 124:2246-2259 (2014), and cryopreserved. Peripheral blood mononuclear cells(PBMC) were obtained by leukapheresis, prepared over a Ficoll-Hypaquegradient (LSMTM; MP Biomedicals, Santa Ana, Calif.), and cryopreserveduntil analysis. Melanoma cell lines were established from enzymaticallyseparated tumor cells cultured in RPMI 1640 supplemented with 10% FBS(HyClone Defined, Logan, Utah) at 37° C. and 5% CO₂. Melanoma cell lineswere mycoplasma negative, and were authenticated based on theidentification of patient-specific somatic mutations, and HLA molecules.

TABLE 10 Months from end of % PD-1+ Cancer last therapy to (of CD8+) #putative Mutations Patient type Prior therapy leukapheresis (mo) PBMCmutations^(d) evaluated^(e) 3713 Mel^(a) IL-2, anti-CTLA-4 7 mo 4.1%4359  7 minimal epitopes 3998 Mel No treatment — 1.9% 279 115 (TMG#1- 7)3784 Mel Surgery, IFN 14 mo  2.1% 440 140 (TMG1-9) 3903 Mel Surgery,MART-F5 55 mo  3.4% 414 308 TCR^(b) (TMG#1- 26) 3926 Mel IL-2, surgery,8 mo 7.4% 346 128 chemo.^(c) (TMG#1- 11) 3759 Mel Surgery, IFN 1 mo 1.0%n.d.^(f) n.e.^(g) 3992 Mel Anti-PD-1, anti- 5 mo   8% n.d. n.e. CTLA-4^(a)Melanoma; ^(b)Adoptive transfer of autologous T cells that weregene-engineered to express a MART-1 HLA-A*0201-restricted T-cellreceptor. ^(c)Chemotherapy patient 3926: dacarbazine and vinblastine.^(d)Putative non-synonymous mutations were defined by: >2 exome variantreads, ≥10% variant frequency in the exome, ≥10 normal reads, andtumor/normal variant frequency ≥5. Common single nucleotidepolymorphisms were filtered. ^(e)Mutations evaluated were selected basedon whole-exome and transcriptome analysis. ^(f)Not deteremined. ^(g)Notevaluated.

TABLE 11 Variable/trait Total (%) Total no. patients 18 Sex Male 14 (78)Female 4 (22) Age 31-40 4 (22) 41-50 3 (17) 51-60 9 (50) 61-70 2 (11)Prior Treatment Surgery 17 (94) Chemotherapy 2 (11) Radiotherapy 2 (11)Immunotherapy 12 (67) Any 2 or more 13 (72) Any 3 or more 8 (44) Notreatment 1 (5)Exome and RNA sequencing.

Tumor biopsies and normal PBMC were subjected to DNA extraction, libraryconstruction, exome capture of approximately 20,000 coding genes, andnext-generation sequencing by Macrogen (Rockville, Md.), Personal GenomeDiagnostics (PGDX, Baltimore, Md.), or the Broad Institute (Cambridge,Mass.). The average number of distinct high quality sequences at eachbase ranged between 100 and 150 for the individual exome libraries.Alignments and variant calling were performed, as described in Tran etal., Science, 344: 641-645 (2014). The total number of putativenon-synonymous mutations (Table 10) was determined using filtersconsisting of >2 exome variant reads, ≥10% variant allele frequency(VAF) in the tumor exome, >10 normal reads, tumor/normal variantfrequency ≥5, and filtering out single nucleotide polymorphisms in dbSNPbuild 138. An mRNA sequencing library was also prepared from a tumorbiopsy using Illumina TRUSEQ RNA library prep kit. RNA alignment wasperformed using STAR (Dobin et al., Bioinformatics, 29: 15-21 (2013))duplicates, were marked using picard's MARKDUPLICATE tools, and FPKMvalues were calculated using cufflinks (Trapnell et al., NatureBiotechnol., 8: 511-515 (2010)). The levels of transcripts encodingputative non-synonymous variants, calculated as fragments per kilobaseper million mapped reads (FPKM), were used to assess expression ofcandidate mutations identified using whole exome data.

The following criteria were used to prioritize mutations forimmunological screening (Table 12). Initially, mutations with a variantallele frequency (VAF)>10% in the tumor exome, as well as mutations thatwere identified in both transcriptome and exome analysis without anyadditional filters, were selected. For some samples (NCI-3903), themutations selected based on exome only were prioritized by selectingthose with >10 variant reads to increase the confidence of mutationcalling. For each of the immunogenic antigens detected, the amino acidchanges are specified.

TABLE 12 Mutation WT Mut AA Patient TMG# Type Gene AA AA positionWt 25-mer Mut 25-mer 3998 TMG1 SNV MAGEA6 E K 168DSLQLVFGIELMEVDPIGHVYIFAT DSLQLVFGIELMKVDPIGHVYIFAT (SEQ ID NO: 80)(SEQ ID NO: 77) 3998 TMG3 SNV PDS5A Y F 1000 MATEKLLSLLPEYVVPYMIHLLAHDMATEKLLSLLPEFVVPYMIYLLAHD PDFTRSQ PDFTRSQ (SEQ ID NO: 81)(SEQ ID NO: 78) H Y 1007 3998 TMG5 SNV MED13 P S 1691PHIKSTVSVQIIPCQYLLQPVKHED PHIKSTVSVQIISCQYLLQPVKHED (SEQ ID NO: 82)(SEQ ID NO: 79)Antibodies, and Phenotypic Characterization of T Cells.

Fluorescently labeled antibodies were purchased from BD Biosciences, SanJose, Calif. (UCHT1, 1.6:100, CD3 PE-CF594; SK7, 1:100, CD3 APC-Cy7;SK1, 0.5:100, CD8 PE-Cy7; 4B4-1, 1.25:100, CD137 APC; NK-1, 3:100, CD57FITC; J168-540, 1.2:100, BTLA PE), eBioscience, San Diego, Calif.(H57-597, 0.5:100, mTCRB FITC; 0323, 2:100, CD27 BV605), Biolegend, SanDiego, Calif. (EH12.2H7, 0.7:100, PD-1 BV421), R&D Systems, Minneapolis,Minn. (344823, 2.6:100, TIM-3 PE and APC), Enzo Life Sciences,Farmingdale, N.Y. (17B4. 1:100, LAG-3 FITC) and Miltenyi Biotec (4B4-1,2.6:100, 4-1BB PE). Anti-PD-1 antibody was from Amplimmune(Gaithersburg, Md., AMP-514, 1/300, PD-1 Alexa Fluor 647). Cell-sortingexperiments were carried out using anti-PD-1 AMP-514 antibody.

To perform the phenotypic characterization, PBMC and tumor single cellsuspensions were thawed into T-cell media (1:1 mix of AIMV media [LifeTechnologies, Waltham, Mass.] and RPMI 1640 media [Lonza, Walkersville,Md.], 5% in-house human serum, 100 U/ml penicillin and 100 μg/mlstreptomycin [Life Technologies], 2 mM L-glutamine [Life Technologies],10 μg/ml gentamicin [Quality Biological Inc., Gaithersburg, Md.], 12.5mM HEPES [Life Technologies]) supplemented with DNAse (Genentech Inc.San Francisco, Calif., 1:1000), centrifuged, and plated at 2e6cells/well in a 24-well plate in the absence of cytokines. After restingthe cells overnight at 37° C. and 5% CO₂, cells were harvested, and 2e6cells were resuspended in 50 μl of staining buffer (PBS, 0.5% BSA, 2 mMEDTA) containing antibodies. Cells were incubated for 30 minutes at 4°C. and washed twice prior to acquisition. Flow cytometry acquisition wascarried out on a modified FORTESSA analyzer, equipped to detect 18fluorescence parameters, or CANTO II flow cytometers (BD Biosciences).Flow cytometry data were analyzed using FLOWJO software (Ashland,Oreg.). Data were gated on live cells (PI negative), single cells. Gateswere set based on fluorescence minus one (FMO) controls.

T-Cell Sorting and In Vitro Expansion.

Cell-sorting was carried out using the BD JAZZ cell sorter (BDBiosciences). For all experiments requiring cell-sorting from PBMC, CD8⁺cells were first enriched using CD8 microbeads (Miltenyi), and stainedas described above in “Antibodies, and phenotypic characterization of Tcells.” When sorting T cells from fresh tumor single cell suspensions,this pre-enrichment step was not performed. Cells were gated on live (PInegative), single cells, CD3⁺, and CD8⁺ cells, and on the population ofinterest. Half of the T cells isolated were spun and snap frozen toperform TRB deep sequencing, and the other half were expanded in vitro.T-cell yields ranged from 3×10³ to 3×10⁵. A similar sorting strategy wasused to sort the 4-1BB⁺ lymphocytes, following a 20 h co-culture.

T cells were expanded in vitro using an excess of irradiated allogeneicfeeders cells (5000 rad) pooled from three donors in T-cell mediasupplemented with 30 ng/ml anti-CD3 (OKT3, Miltenyi Biotec) and 3000 IUof IL-2 (Aldesleukin, Chiron). After day 6, half of the media wasreplaced with fresh T-cell media containing IL-2 every other day. At day15, T cells were either used in co-culture assays or cryopreserved,until future analysis. Of note, enrichment of mutation-specific T cellswas consistent between replicate CD8³⁰PD-1⁺ T cell cultures, butstochastic outgrowth or loss of T cell reactivities can be observed andbecome more apparent when starting with less than 3×10³ CD8⁺PD-1⁺ Tcells. The minimum material required to sort 3×10³ CD8⁺PD-1⁺ cells isapproximately 1×10⁷ PBMC.

Generation of Autologous Antigen Presenting Cells (APCs).

Immature dendritic cells (CD11c⁺, CD14⁻, CD80^(low), CD86⁺ and HLA-DR⁺)were generated from PBMC using the plastic adherence method, asdescribed in Tran et al., Science, 344: 641-645 (2014). On day 3, DCmedia was added, and at day 5-6 DCs were harvested and used inelectroporation experiments or cryopreserved. DC media comprised of RPMIsupplemented with 5% human serum, 100 U/ml penicillin and 100 μg/mlstreptomycin, 2 mM L-glutamine (Life Technologies), 800 IU/ml GM-CSF and200 U/ml IL-4 (Peprotech, Rocky Hill, N.J.). When used aftercryopreservation, cells were thawed into DC media, spun at 1000 RPM for10 min, resuspended in DC media at 2×10⁶ cells/ml, and incubated at 37°C. and 5% CO₂ for 2 h, prior to electroporation or peptide pulsing.

Autologous B cells were isolated from autologous PBMC by positiveselection using CD19⁺ microbeads (Miltenyi Biotec) and expanded usingirradiated NIH3T3 CD40L cells and IL-4 (Peprotech), as described in Tranet al., Science, 344: 641-645 (2014). B cells were harvested at day 5-6after the initial stimulation, and either re-stimulated, cryopreserved,or used in co-culture assays. When used after cryopreservation, B cellswere thawed into B cell media 16-24 h prior to using them in co-cultureassays. B cell media comprised of IMDM media (Quality Biological Inc.,Gaithersburg, Md.) supplemented with 10% human serum, 100 U/mlpenicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, and 200 U/mlIL-4 (Peprotech, Rocky Hill, N.J.).

Construction of TMGs, and In Vitro Transcription of TMG RNA.

Tandem minigenes (TMGs) were constructed as described in Lu et al.,Clin. Cancer Res., 20: 3401-3410 (2014); Tran et al., Science, 344:641-645 (2014). Briefly, a minigene was constructed for eachnon-synonymous variant identified, composed of the mutated amino acidflanked by 12 amino acids of the wild-type protein sequence. Up to 16minigenes were strung together to generate a tandem minigene (TMG)construct. These TMG constructs were codon optimized and cloned in frameinto pcRNA2SL using EcoRI and BamHI. pcRNA2SL is based on the pcDNA3.1,and was modified to include a signal sequence and a DC-LAMP traffickingsequence to enhance processing and presentation (Wu et al., PNAS, 92:11671-11675 (1995)). The sequences were verified by ranger sequencing.Following linearization of the constructs, phenol chloroform extractionwas performed and DNA was precipitated with sodium acetate and ethanol.Next, 1 μg of linearized DNA was used to generate in vitro transcribedRNA using the MMESSAGE MMACHINE T7 Ultra kit (Life Technologies), asinstructed by the manufacturer. RNA was precipitated using LiCl₂, andresuspended at 1 μg/μl. To screen for recognition of cancer germlineantigens NY-ESO-1, MAGEA3 and SSX2, and melanoma differentiationantigens MART1, GP100 and TYROSINASE, full-length amino acid sequenceswere cloned individually into pcRNA2SL using EcoRI and BamHI, and theseconstructs were used to generate IVT RNA.

Transfection of RNA or DNA.

DCs were resuspended in Opti-MEM (Life Technologies) at 10-40×10⁶cells/ml. 8 μg of IVT RNA was aliquoted into the bottom of a 2 mm gapelectroporation cuvette, and 100 μl of DCs were added. DCs wereelectroporated with 150 V, 10 ms, and 1 pulse, using a BTX-830 squarewave electroporator (Holliston, Mass.). Cells were gently resuspendedinto DC media and transferred into ultra-low attachment polysterene24-well plate (corning) at approximately 1×10⁶ DCs/ml, and restedovernight at 37° C., 5% CO₂. Transfection efficiencies were routinelybetween 70-90% assessed with a control GFP RNA (not shown). Inco-culture assays, the irrelevant TMG RNA control was a random TMG froma different patient.

HLA alleles were cloned into pcDNA3.1. To interrogate which HLA allelespresented the neo-antigens identified, COST cells were co-transfectedwith TMG DNA constructs and plasmids encoding the individual HLAmolecules using LIPOFECTAMINE 2000 reagent (Life Technologies). After 16h, cells were harvested and used as targets in co-culture assays.

HLA-I Alleles, Peptide Prediction and Pulsing.

HLA was determined from next generation sequencing data using thealgorithm PHLAT (Bai et al., BMC Genomics, 15: 325 (2014)). (NCI-3713:HLA-A*02:01, A*29:02, B*44:03, B*51:01, C*15:02, C*16:01. NCI-3998:HLA-A*01:01, A*30:02, B*15:01, B*18:01, C*03:03, C*05:01. NCI-3784:HLA-A*01:01, A*03:01, B*07:02, C*07:02. NCI-3903: HLA-A*02:01, A*24:02,B*27:02, B*38:01, C*02:02, C*12:03. NCI-3926: HLA-A*01:01, A*02:01,B*08:01, B*13:02, C*06:02, C*07:01).

Candidate 8 to 11-mers containing the mutated residues that werepredicted to bind with high affinity to the patients' HLA-I moleculeswere identified using the immune epitope database (IEDB) (Vita et al.,Nucleic Acids Res., 43: D405-412 (2015)). Crude and HPLC peptides weresynthesized by GenScript (Piscataway, N.J.), and resuspended in DMSO at10 mg/ml and stored at −20° C.

For experiments requiring peptide pulsing, DCs or B cells wereresuspended in DC or B cell media, respectively, at 1e6 cells/ml. DCswere incubated overnight at 37° C. and 5% CO₂ with wild-type or mutated25-mers at a concentration of 10 μg/ml in DC media. B cells were pulsedwith 1 μg/ml or with 10-fold serial dilutions of minimal epitopesstarting at 10 μg/ml for 2 h at 37° C. and 5% CO₂. DCs or B cells werewashed once with PBS prior to co-incubation with T cells.

Co-Culture Assays: IFN-γ ELISPOT, and Flow Cytometry Detection ofActivation Marker 4-1BB.

Both IFN-γ enzyme-linked immunospot assay (ELISPOT) and 4-1BBupregulation at 20 h after the co-culture were used to measure targetcell recognition by T cells. After 15 days of T-cell expansion, orfollowing overnight rest of cryopreserved T cells in T cell mediasupplemented with 3000 IU/ml IL-2, T cells were washed to remove excesscytokines. In the ELISPOT assays, 2×10⁴ T cells were added per well in a96-well plate. When DCs electroporated with IVT RNA encoding for TMGs orshared antigens were used as targets, approximately 3-7×10⁴ cells/wellwere used in a 96-well plate. When peptide-pulsed B cells were used,8×10⁴ to 1.5×10⁵ cells were added per well. All co-cultures were carriedout in T-cell media in the absence of exogenously added cytokines. Tcells cultured alone or stimulated with plate bound anti-CD3 (OKT3) wereused as controls in all the assays. CEF RNA encoding for epitopesderived from CMV, EBV, and Flu (CEF) were included as controls in allthe immunological screening assays (Nielsen et al., J. Immunol., Meth.,360: 149-156 (2010)).

IFN-γ ELISPOT assays were carried out as described in Tran et al.,Science, 344: 641-645 (2014). The raw data were plotted withoutsubtracting the background. Greater than 40 spots, and greater thantwice the background was considered positive T cell reactivity. Prior toprocessing the ELISPOT assay, cells were harvested for flow-cytometrydetection of 4-1BB upregulation, as described in Tran et al., Science,344: 641-645 (2014).

TCR Deep Sequencing and Analysis.

TCR-α (TRA) and TCR-β (TRB) deep sequencing were performed on genomicDNA by Adaptive Biotechnologies (Seattle, Wash.). For the enrichedpopulations of TMG-reactive cells, DNA was extracted from 1 e6lymphocytes. The number of circulating and tumor-resident CD8⁺lymphocytes that were sequenced ranged from 3×10³ to 3×10⁵. The coverageper sample was >10×. Only productive TCR rearrangements were used in thecalculations of TCR frequencies and TRB overlap. Analysis of TRB overlapof CDR3 nucleotide sequences between two given populations wascalculated using an IMMUNOSEQ analyzer (Adaptive Biotechnologies,Seattle, Wash.) using the following formula: sample TRB overlap=[sharedsequence reads in A+shared sequence reads in B]/Σsequence reads in A+B).Weighing in the frequency of the shared sequences rather than the totalnumber of shared sequences helped account for potentially differentsized samples. A TRB overlap of 1 represents 100% overlap between twopopulations.

Retroviral Vector Construction, Production and Transduction of T cells.

For NCI-3998, TCRs were constructed by pairing the dominant TRA and TRBchains, and for each population the TCRs were designated based on therank of the TRA and TRB (TCR A rank #/B rank #) within the populationsequenced. In total, 2 TCRs were assembled from the TMG1(MAGEA6_(E>K))-reactive population (TCR A1/B1, TCR A1/B2), and 4 TCRsfrom the TMG3 (PDS5A_(Y>F; H>Y))-reactive, as well as the TMG5(MED13_(P>S))-reactive populations (TCR A1/B1, TCR A1/B2, TCR A2B1, TCRA2/B2). Briefly, TRA V-J regions and TRB V-D-J regions were fused to themouse constant TCR-alpha and beta chains (Cohen et al., Cancer Res., 66:8878-8886 (2006)), respectively. Mouse constant regions were modified,as described in (Cohen et al., Cancer Res., 67: 3898-3903 (2007);Haga-Friedman et al., J. Immunol., 188: 5538-5546 (2012). Thefull-length TCRB and TCRA chains were cloned, in this orientation, intopMSGV1 retroviral vector separated by a furin SGSG P2A linker(GenScript). For all TCRs, the amino acid residue at position 2 of thebeta chain was changed from a glycine to an alanine in order tofacilitate cloning into the vector.

Transient retroviral supernatants were generated, and autologous PBMCswere transduced as described in Tran et al., Science, 344: 641-645(2014). Transduced T cells were used at day 15 or cryopreserved untilused. GFP and mock transduced T cells were used as controls in alltransduction experiments.

Statistical Analysis.

Data were reported as the median, mean±SEM, or mean±SD, as specified.The Mann-Whitney test was used to compare the percentage of PD-1expression between PBMC and fresh tumor single cell suspensions. Dunn'stest for multiple comparisons was used to analyze the statisticaldifferences in TRB overlap. Statistical analysis was carried out usingPRISM program 6.0 (GRAPHPAD Software Inc., La Jolla, Calif.). Unlessotherwise specified, experiments were performed without duplicates. Alldata are representative of at least 2 experiments.

Example 11

This example demonstrates the expression of PD-1 on peripheral blood andtumor-infiltrating CD8⁺ T cells in patients with melanoma.

The expression of PD-1 on peripheral blood and tumor-infiltrating CD8⁺ Tcells was compared. PD-1 expression accounted for approximately 36% ofthe CD8⁺ TIL population, but matched peripheral blood samples from thesame individuals contained only a median of 4.1% CD8⁺PD-1⁺ cells.Moreover, circulating CD8⁺ lymphocytes had limited co-expression of theinhibitory and co-stimulatory cell surface receptors PD-1, TIM-3, LAG-3and 4-1BB compared to tumor-resident CD8⁺ lymphocytes. Thus, fewPD-1-expressing circulating CD8⁺ lymphocytes are present in patientswith melanoma.

Example 12

This example demonstrates the screening of circulating in vitro expandedCD8⁺ cells from melanoma patients for recognition of mutations.

It was next examined whether selection of circulating CD8+PD-1+lymphocytes was able to prospectively identify neoantigen-specific CD8+T cells in the blood of four individuals with melanoma. Ahigh-throughput personalized screening strategy capable of evaluating Tcell reactivity to neoantigens presented on all of the HLA restrictionelements of the individual was used. Briefly, mutations selected on thebasis of tumor-exome and transcriptome analyses were incorporated intooligonucleotides (minigenes) that encoded a 25-residue peptide (25-mer),and these oligonucleotides were then concatenated to yield tandemminigenes (TMGs; designated in numerical order and for each patient).Each TMG encoded up to 16 minigenes, and the requisite number of TMGsthat allowed for the expression of all of the mutant 25-mers that wereidentified were constructed.

In parallel, CD8⁺ lymphocytes were separated from pre-treatmentperipheral blood mononuclear cells (PBMCs) into CD8⁺, CD8⁺PD-1⁻,CD8⁺PD-1⁺, and CD8⁺PD-1^(hi) (defined as the top 20% of PD-1-expressingCD8⁺ T cells), and expanded for 15 days. In vitro transcribed TMG RNAwas electroporated into immature autologous dendritic cells (DCs) thatwere employed as targets in a T cell co-culture assay. Using thisapproach, the circulating in vitro expanded CD8⁺ subsets from 4individuals with metastatic melanoma (patients NCI-3998, NCI-3784,NCI-3903, and NCI-3926, see Table 10) were screened for recognition of115, 140, 308, and 128 mutations, respectively.

Example 13

This example demonstrates the detection of mutation-reactive lymphocyteswithin the CD8⁺PD-1⁺ subset of Example 12.

Although the unseparated peripheral blood CD8+ cells, as well as theCD8+PD-1− lymphocytes, from NCI-3998 showed limited recognition of themutant 25-mers encoded by TMG1 (hereafter referred to as recognition ofTMG1 or TMG1 reactive), the circulating CD8+PD-1+ lymphocyte subsetshowed enhanced TMG1 reactivity and low, but reproducible, reactivity toTMG3 and TMG5. Based on upregulation of the activation marker 4-1BB, thefrequency of CD8+PD-1+ cells that were reactive to DCs expressing theseTMG-encoded peptides was 1.8% for TMG1, 0.5% for TMG3 and 0.3% for TMG5.Additionally, recognition of TMG1 and TMG3 by the CD8+PD-1^(hi) subsetwas also observed. Similarly, CD8+PD-1+ and CD8+PD-1^(hi), but not CD8+or CD8+PD-1+, lymphocytes from the peripheral blood of subjects NCI-3784and NCI-3903 showed T cell reactivity to neoantigens. CirculatingCD8⁺PD-1⁺ cells from NCI-3784 recognized at least three neo-antigensencoded by TMG3, TMG5 and TMG8, and peripheral blood CD8⁺PD-1⁺lymphocytes isolated from NCI-3903 detected at least one neo-antigenexpressed by TMG9. NCI-3926 peripheral blood lymphocytes did not showT-cell reactivity to any of the neo-antigens screened. Overall,circulating mutation-reactive lymphocytes were prospectively identifiedin 3 of 4 melanoma patients evaluated, and these cells were consistentlydetected within the CD8⁺PD-1⁺ lymphocytes. Notably, with the exceptionof NCI-3998, who displayed low level recognition of TMG1 in theunseparated population of circulating CD8⁺ T cells, selection ofCD8⁺PD-1⁺ or PD-1^(hi) lymphocytes from the blood of the patients wasnecessary to expose CD8+ T cell reactivity to neoantigens.

Example 14

This example demonstrates the isolation of TCRs from themutation-reactive lymphocytes of Example 13.

The specific neo-antigens targeted by the mutation-reactive lymphocyteswere next analyzed. Given the low frequency of some of the reactivities,and the polyclonal nature of the circulating PD-1⁺ subset, TMG-reactivecells were enriched by selecting 4-1BB⁺ lymphocytes following aco-culture with specific TMGs, expanding them in vitro, andco-incubating them with DCs individually pulsed with the mutated 25-mersencoded by the corresponding TMG (Table 12). In a representativeexample, TMG1−, TMG3− and TMG5− reactive cells isolated from thecirculating CD8+PD-1+ subset of subject NCI-3998 showed reactivity toneoantigens derived from mutations in the genes MAGE family member A6(MAGEA6), PDS5 cohesion-associated factor A (PDS5A) and mediator complexsubunit 13 (MED13) (which are referred to as MAGEA6_(E>K),PDS5A_(Y>F;H>Y) and MED13_(P>S), respectively). The minimal predictedepitopes were determined, synthesized, and tested, and the TMG-reactivecells demonstrated specific recognition of the mutated neo-epitopes overthe wild-type counterparts. The HLA alleles presenting the neo-antigenswere also identified. Although MAGEA6_(E>K) and PDS5A_(Y>F;H>Y) werepresented by the alleles encoding HLA-A*30:02 and HLA-C*03:03,respectively, recognition of the MED13_(P>S) neo-epitope was restrictedto alleles encoding HLA-A*30:02 and HLA-B*15:01. Deep-sequencinganalyses of the variable V-J or V-D-J region of the TRA and TRB genes(which encode the hypervariable regions of the TCR-α and TCR-β chainsthat are important for peptide recognition by the TCR) of the enrichedpopulations of neoantigen-specific CD8+ T cells revealed multipledominant TRA and TRB sequences that were unique for each of the T cellpopulations. To study the specificity of the mutation-specific cells atthe clonal level, TCRs were constructed by pairing the sequencesencoding the 2 most dominant TRA and TRB CDR3 sequences (Linnemann etal., Nature Med., 19: 1534-1541 (2013)) from the MAGEA6_(E>K),PDS5A_(Y>F;H>Y), or the MED13_(P>S) neo-antigen specific lymphocytes,and cloning them into retroviral vectors used to transduce autologousPBMCs. The full-length alpha and beta chain amino acid sequences encodedby the vectors are shown in Table 13. The two TCRs constructed bypairing the most dominant and the second most dominant TRA and TRBsequences (which are referred to as TCR A1/B1 and TCR A2/B2) from theMAGEA6_(E>K)-reactive population displayed MAGEA6_(E>K) recognition,based on 4-1BB upregulation against the mutated MAGEA6_(E>K) minimalepitope. Four TCRs (TCR A1/B1, TCR A1/B2, TCR A2/B1, TCR A2/B2) wereassembled for each of the remaining MED13_(P>S) andPDS5A_(Y>F;H>Y)-specific lymphocyte populations. Two of the fourpotential MED13_(P>S)-specific TCR-expressing lymphocytes tested,TCRA1/B1 and TCRA2/B2, recognized the MED13_(P>S) mutated 25-mer peptideand recognition of MED13_(P>S) was restricted to HLA-B*15:01 andHLA-A*30:02, respectively. Finally, out of four PDS5A_(Y>F;H>Y) TCRsconstructed and screened, one single TCR displayed specific recognitionof TMG3 and the PDS5A_(Y>F;H>Y) neo-epitope.

TABLE 13 TRA rank/TRB rank (T-cell population TCR alpha chain TCR betachain Reactivity of origin) TRAV/TRAJ sequence TRBV/TRBJ sequenceMAGEA6^(E168K) A1/B1 TRAV21*01/ SEQ ID NO: 51 TRBV7-3*01/ SEQ ID NO: 52(TMG1 enriched) TRAJ21*01F TRBJ1-2*01 MAGEA6^(E168K) A2/B2 TRAV39*01/SEQ ID NO: 53 TRBV7-6*01/ SEQ ID NO: 54 (TMG1 enriched) TRAJ58*01TRBJ1-2*01 PDS5A_(Y1000F; H1007Y) A1/B2 TRAV38-1*01/ SEQ ID NO: 55TRBV27*01/ SEQ ID NO: 56 (TMG3-enriched) TRAJ53*01 TRBJ2-2*01MED13_(P1691S) A1/B1 TRAV12-1*01/ SEQ ID NO: 57 TRBV9*01/ SEQ ID NO: 58(TMG5-enriched) TRAJ27*01 TRBJ2-1*01 MED13_(P1691S) A2/B2 TRAV12-2*01/SEQ ID NO: 59 TRBV27*01/ SEQ ID NO: 60 (TMG5-enriched) TRAJ29*01TRBJ2-7*01

In NCI-3784, peripheral blood neo-antigen specific responses wereidentified for three mutated antigens FLNA_(R>C), KIF16B_(L>P), andSON_(R>C) presented by HLA-B*07:02. Moreover, circulating CD8⁺PD-1⁺lymphocytes reactive against TMG9 from NCI-3903 displayedmutation-specific recognition of KIF1BP_(P>S) 8-mer presented byHLA-B*38:01, and this population contained 3 dominant TRB clonotypes.Thus, selection of circulating CD8⁺PD-1⁺ lymphocytes led to theprospective identification of a diverse mutation-specific T-cellresponse in 3 of 4 melanoma patients tested, with 3, 3, and 1 unique,patient-specific neo-antigens recognized, respectively.

Example 15

This example demonstrates that selection of circulating CD8+PD-1+lymphocytes reveals that the T-cell response to mutated antigens derivedfrom TIL also existed in the blood of Patient 3713 prior to TIL therapy.

Patient NCI-3713 experienced a complete tumor regression followingadministration of TIL-3713. Previous studies showed that TIL-3713derived from a lung metastasis recognized multiple mutated neo-epitopesincluding WDR46_(T>I), SRPX_(P>L), AFMID_(A>V), HELZ2_(D>N),CENPL_(P>L), AHNAK_(S>F), and PRDX3_(P>L). Analysis of the pre-treatmentPBMCs from this patient demonstrated recognition of 6 of 7 neo-epitopestested (recognizing WDR46_(T>I), SRPX_(P>L), AFMID_(A>V), HELZ2_(D>N),CENPL_(P>L), and PRDX3_(P>L), but not AHNAK_(S>F)). Reactivity wasuniquely identified within the circulating CD8⁺PD-1⁺ and CD8+PD-1^(hi),but not the CD8⁺ or the CD8⁺PD-1⁺ lymphocytes. T-cell reactivitiesobserved were mutation-specific, as they displayed preferentialrecognition of the mutated over the wild-type peptides, and thepercentage of neo-antigen-specific cells based on 4-1BB up-regulationranged from 0.5% to up to 21% of the CD8⁺PD-1^(hi) cells. Thus,selection of circulating CD8⁺PD-1⁺ lymphocytes revealed that the T-cellresponse to mutated antigens derived from TIL also existed in the bloodof this patient prior to TIL therapy.

Example 16

This example demonstrates the recognition of autologous tumor by theenriched populations of mutation-specific T cells and T-cell receptorsisolated in Example 14.

In view of their potential use to treat cancer, the recognition ofautologous tumor by the enriched populations of mutation-specific Tcells and T-cell receptors isolated was next examined. MAGEA6_(E>K),PDS5A_(Y>F;H>Y), or the MED13_(P>S) TCR-transduced T cells fromNCI-3998, and mutation-specific CD8⁺ T cells derived from the blood ofNCI-3784, and 3903 recognized their corresponding autologous tumor cellline at variable levels (FIG. 2A), either with or without pre-treatmentof the autologous tumor cell lines with IFN-γ, which can enhanceprocessing and presentation of epitopes on HLA molecules. Furthermore,in all 5 individuals studied, the circulating CD8⁺PD-1⁺, but not theCD8⁺PD-1⁻ cells, displayed direct tumor recognition, as evidenced bydetection of 4-1BB up-regulation (FIG. 2B) and IFN-γ release. Thefrequency of tumor-reactive cells within the circulating CD8⁺PD-1⁺lymphocytes ranged from 6.3% to 24.6%. Circulating CD8⁺PD-1⁺ cells fromNCI-3926 did not recognize any of the mutated antigens tested, butrecognized autologous tumor. Additionally, the percentage oftumor-reactive CD8⁺PD-1⁺ lymphocytes from NCI-3998 and 3784 (9.5%, and24.6%, respectively) exceeded that observed against the neo-antigensevaluated, suggesting that either additional neo-antigens or non-mutatedtumor antigens may be recognized by the circulating CD8⁺PD-1⁺ subset.Indeed, in all four patients evaluated, the circulating CD8⁺PD-1⁺ and orCD8⁺PD-1^(hi) cells also displayed recognition of one or more cancergermline antigens or melanoma differentiation antigens tested, includingNY-ESO-1, MAGEA3, SSX2, MART1, GP100 and TYR. While the peripheral bloodCD8⁺PD-1⁺ T cells from NCI-3903 recognized SSX2, circulating CD8⁺PD-1⁺T-cell subsets derived from NCI-3926 and NCI-3998 recognized NY-ESO-1,and the CD8⁺PD-1^(hi) lymphocytes from NCI-3784 displayed reactivityagainst MAGEA3, and GP100. MART1 and TYR were not recognized by any ofthe CD8⁺ T-cell subsets tested. The relative frequency of circulatingCD8⁺PD-1⁺ T cells targeting mutated antigens and self-antigens washighly variable from patient to patient. The relative frequency ofcirculating CD8⁺PD-1⁺ T cells targeting mutated antigens andself-antigens for representative Patient 3998 is shown in Table 14.

TABLE 14 Peripheral blood Tumor CD8+PD-1+ CD8+PD-1hi CD8+PD-1+ % oftotal % of total % of total reactivities reactivities reactivities %4-1BB+ detected % 4-1BB+ detected % 4-1BB+ detected MAGEA6_(E168K)(TMG1) 2.4 10.0 2.9  8.8 3.8 30.1 PDS5A_(Y1000F; H1007Y) 0.6  2.5 0.5 1.5 0.2  1.6 (TMG3) 0.3  1.3 N.D. N.D. 0.9  7.4 MED13_(P1691S) (TMG5)3.3 13.8 3.4 10.3 4.9 40.2 Mutated antigens 20.7  86.2 29.7  89.7 7.359.8 NY-ESO-1 20.7  86.2 29.7  89.7 7.3 59.8 Self-antigens 3998mel 9.57.2 11.2

Example 17

This example demonstrates the characteristics of the CD8⁺PD-1⁺lymphocytes of Examples 11-16.

The findings in Examples 11-16 indicated that circulating CD8⁺PD-1⁺lymphocytes were enriched in cancer mutation-specific cells as well asother tumor-specific T cells. Additionally, simultaneous screening ofmatched circulating and tumor-resident CD8⁺PD-1⁺ lymphocytes in 4patients revealed a high degree of similarity in the tumor antigenstargeted by both populations. In concordance, TRB deep sequencing ofmatched tumor-resident and circulating lymphocytes in the absence of invitro expansion manifested a relatively high degree of overlap betweenTRB repertoires of the tumor-infiltrating and circulating CD8⁺PD-1⁺subsets, but far less with the circulating CD8⁺ or CD8⁺PD-1⁻ (FIG. 2C).The specific antigens recognized by the circulating CD8⁺PD-1⁺lymphocytes and the TIL infusion product these patients received werealso similar.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An isolated or purified nucleic acidcomprising a nucleotide sequence encoding a TCR, or an antigen-bindingportion thereof, wherein the TCR, and the antigen-binding portionthereof, comprise the amino acid sequences of: (a) SEQ ID NOs: 5-10 or(b) SEQ ID NOs: 13-18.
 2. The nucleic acid according to claim 1, whereinthe TCR, and the antigen-binding portion thereof, comprise the aminoacid sequences of: (a) SEQ ID NOs: 11-12 or (b) SEQ ID NOs: 19-20. 3.The nucleic acid according to claim 1, wherein the TCR, and theantigen-binding portion thereof, further comprise the amino acidsequences of: (a) SEQ ID NO: 45, wherein (i) X at position 48 is Thr orCys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe,Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile,Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Met, or Trp; and (b) SEQ ID NO: 46, wherein X at position57 is Ser or Cys.
 4. The nucleic acid according to claim 1, wherein theTCR, and the antigen-binding portion thereof, comprise the amino acidsequences of: (a) SEQ ID NOs: 51-52 or (b) SEQ ID NOs: 53-54.
 5. Anisolated or purified nucleic acid comprising a nucleotide sequenceencoding a polypeptide, wherein the polypeptide comprises the amino acidsequences of: (a) SEQ ID NOs: 5-10 or (b) SEQ ID NOs: 13-18.
 6. Thenucleic acid according to claim 5, wherein the polypeptide comprises theamino acid sequence of: (a) SEQ ID NOs: 11-12 or (b) SEQ ID NOs: 19-20.7. The nucleic acid according to claim 5, wherein the polypeptidefurther comprises the amino acid sequences of: (a) SEQ ID NO: 45,wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 isSer, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) Xat position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and(b) SEQ ID NO: 46, wherein X at position 57 is Ser or Cys.
 8. Thenucleic acid according to claim 5, wherein the polypeptide comprises theamino acid sequences of: (a) SEQ ID NOs: 51-52 or (b) SEQ ID NOs: 53-54.9. An isolated or purified nucleic acid comprising a nucleotide sequenceencoding a protein, wherein the protein comprises: (a) a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs: 5-7and a second polypeptide chain comprising the amino acid sequences ofSEQ ID NOs: 8-10 or (b) a first polypeptide chain comprising the aminoacid sequences of SEQ ID NOs: 13-15 and a second polypeptide chaincomprising the amino acid sequences of SEQ ID NOs: 16-18.
 10. Thenucleic acid of claim 9, wherein the protein comprises: (a) a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 11and a second polypeptide chain comprising the amino acid sequence of SEQID NO: 12 or (b) a first polypeptide chain comprising the amino acidsequence of SEQ ID NO: 19 and a second polypeptide chain comprising theamino acid sequence of SEQ ID NO:
 20. 11. The nucleic acid of claim 9,wherein: (a) the first polypeptide chain further comprises the aminoacid sequence of SEQ ID NO: 45, wherein (i) X at position 48 is Thr orCys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe,Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile,Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Met, or Trp; and (b) the second polypeptide chain furthercomprises the amino acid sequence of SEQ ID NO: 46, wherein X atposition 57 is Ser or Cys.
 12. The nucleic acid according to claim 9,wherein the protein comprises: (a) a first polypeptide chain comprisingthe amino acid sequence of SEQ ID NO: 51 and a second polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 52 or (b) a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 53and a second polypeptide chain comprising the amino acid sequence of SEQID NO:
 54. 13. A recombinant expression vector comprising the nucleicacid of claim
 1. 14. A host cell comprising the recombinant expressionvector of claim
 13. 15. A population of cells comprising at least onehost cell of claim
 14. 16. A pharmaceutical composition comprising thepopulation of cells of claim 15 and a pharmaceutically acceptablecarrier.